Methods and compositions for improving soybean yield

ABSTRACT

Provided herein are isolated yield enhancing  Methylobacterium  sp., compositions comprising yield enhancing  Methylobacterium  sp., methods of using the compositions to increase yield of soybean plants, plant parts, and soybean plants derived therefrom, and methods of making the compositions. Such yield enhancing  Methylobacterium  sp. are in certain instances referred to herein as simply “ Methylobacterium ”. In certain embodiments, yield enhancing  Methylobacterium  sp. can be distinguished from other yield neutral or yield negative  Methylobacterium  by assaying the  Methylobacterium  sp. for improved yield in a controlled environment (i.e. a growth chamber or greenhouse) or in a field test in comparison to untreated control plants.

CROSS-REFERENCE TO RELATED APPLICATIONS

This international patent application claims the benefit of U.S. PatentApplication No. 61/911,698, filed Dec. 4, 2013.

BACKGROUND

One-carbon organic compounds such as methane and methanol are foundextensively in nature, and are utilized as carbon sources by bacteriaclassified as methanotrophs and methylotrophs. Methanotrophic bacteriainclude species in the genera Methylobacter, Methylomonas,Methylomicrobium, Methylococcus, Methylosinus, Methylocystis,Methylosphaera, Methylocaldum, and Methylocella (Lidstrom, 2006).Methanotrophs possess the enzyme methane monooxygenase, thatincorporates an atom of oxygen from O₂ into methane, forming methanol.All methanotrophs are obligate one-carbon utilizers that are unable touse compounds containing carbon-carbon bonds. Methylotrophs, on theother hand, can also utilize more complex organic compounds, such asorganic acids, higher alcohols, sugars, and the like. Thus,methylotrophic bacteria are facultative methylotrophs. Methylotrophicbacteria include species in the genera Methylobacterium, Hyphomicrobium,Methylophilus, Methylobacillus, Methylophaga, Aminobacter,Methylorhabdus, Methylopila, Methylosulfonomonas, Marinosulfonomonas,Paracoccus, Xanthobacter, Ancylobacter (also known as Microcyclus),Thiobacillus, Rhodopseudomonas, Rhodobacter, Acetobacter, Bacillus,Mycobacterium, Arthobacter, and Nocardia (Lidstrom, 2006).

Most methylotrophic bacteria of the genus Methylobacterium arepink-pigmented. They are conventionally referred to as PPFM bacteria,being pink-pigmented facultative methylotrophs. Green (2005, 2006)identified twelve validated species in the genus Methylobacterium,specifically M. aminovorans, M. chloromethanicum, M. dichloromethanicum,M. extorquens, M. fujisawaense, M. mesophilicum, M. organophilum, M.radiotolerans, M. rhodesianum, M. rhodinum, M. thiocyanatum, and M.zatmanii. However, M. nidulans is a nitrogen-fixing Methylobacteriumthat is not a PPFM (Sy et al., 2001). Methylobacterium are ubiquitous innature, being found in soil, dust, fresh water, sediments, and leafsurfaces, as well as in industrial and clinical environments (Green,2006).

SUMMARY

Provided herein are isolated yield enhancing Methylobacterium sp.,compositions comprising yield enhancing Methylobacterium sp., methods ofusing the compositions to increase yield of soybean plants, plant parts,and soybean plants derived therefrom, and methods of making thecompositions. Such yield enhancing Methylobacterium sp. are in certaininstances referred to herein as simply “Methylobacterium”. In certainembodiments, yield enhancing Methylobacterium sp. can be distinguishedfrom other yield neutral or yield negative Methylobacterium by assayingthe Methylobacterium sp. for improved yield in a controlled environment(i.e. a growth chamber or greenhouse) or in a field test in comparisonto untreated control plants or in comparison to control plants treatedyield neutral or yield negative Methylobacterium, and combinationsthereof. In certain embodiments, the yield enhancing Methylobacteriumsp. is a Methylobacterium isolate selected from the group consisting ofISO02 (NRRL B-50930), ISO03 (NRRL B-50931), ISO09 (NRRL B-50937), ISO10(NRRL B-50938), and derivatives thereof.

Methods for improving soybean plant yield that comprise applying acomposition comprising a Methylobacterium sp. to a soybean seed, or to asoybean plant or part thereof at a first time of about the VE to aboutthe V4 stage of development; and/or at a second time about the V5 toabout the R6 stage of development are provided herein. In certainembodiments, the composition that is applied comprises: (a) a solidsubstance with the Methylobacterium grown thereon and adhered thereto;or (b) an emulsion having the Methylobacterium grown therein. Alsoprovided are methods for improving soybean plant yield that comprise:(a) applying a composition comprising a Methylobacterium sp. to asoybean seed, or to a soybean plant or part thereof at a first time ofabout the VE to about the V4 stage of development; and/or at a secondtime about the V5 to about the R6 stage of development; wherein thecomposition comprises: (i) a solid substance with the Methylobacteriumgrown thereon and adhered thereto; (ii) an emulsion having theMethylobacterium grown therein; or (iii) a Methylobacterium isolateISO02 (NRRL B-50930), ISO03 (NRRL B-50931), ISO09 (NRRL B-50937), ISO10(NRRL B-50938), or a derivative thereof and an agriculturally acceptableadjuvant, excipient, or combination thereof; and, (b) growing thesoybean plant to maturity, thereby improving yield of the soybean plant.In certain embodiments, the solid substance with the Methylobacteriumgrown thereon and adhered thereto is provided in a liquid or anemulsion. In certain embodiments, the methods further comprise growingthe soybean plant to maturity. In certain embodiments, the compositionis applied in the first time at the VE to V2 stage of development, atthe V2 to the V3 stage of development, at the V3 to V4 stage ofdevelopment, or at about the V3 stage of development. In certainembodiments, the composition is applied at the V6 to the R1 stage ofdevelopment, at the R1 to R2 stage of development, or at about the R1stage of development. In certain embodiments, the composition comprisesa solid substance with the Methylobacterium grown thereon and adheredthereto. In certain embodiments, the composition is a solid compositionthat contains the Methylobacterium sp. at a titer of about 1×10⁶ CFU/gmto about 1×10¹⁴ CFU/gm. In certain embodiments, the composition is aliquid composition containing the solid substance or an emulsion thatcontains the Methylobacterium sp at a titer of about 1×10⁶ CFU/mL toabout 1×10¹¹ CFU/mL. In certain embodiments, the Methylobacterium sp. isISO02 (NRRL B-50930), ISO03 (NRRL B-50931), ISO09 (NRRL B-50937), ISO10(NRRL B-50938), or a derivative thereof. In certain embodiments, theMethylobacterium sp. is ISO09 (NRRL B-50937), ISO10 (NRRL B-50938), or aderivative thereof. In certain embodiments, the composition comprisesthe solid substance or the emulsion and wherein the Methylobacterium sp.is ISO02 (NRRL B-50930), ISO03 (NRRL B-50931), ISO09 (NRRL B-50937),ISO10 (NRRL B-50938), or a derivative thereof. In certain embodiments,the Methylobacterium sp. is a glyphosate resistant or glufosinateresistant derivative of ISO02 (NRRL B-50930), ISO03 (NRRL B-50931),ISO09 (NRRL B-50937), or ISO10 (NRRL B-50938). In certain embodiments,the Methylobacterium sp. is selected for glyphosate resistance, thesoybean plant is a glyphosate tolerant soybean plant, and a formulationcontaining glyphosate is also applied at about the V2 to about the V4stage of soybean plant development. In certain embodiments, theMethylobacterium sp. is ISO09 (NRRL B-50937), ISO10 (NRRL B-50938), or aderivative thereof. In certain embodiments of any of the aforementionedmethods, the applied composition coats or partially coats the soybeanseed or the soybean plant or a part thereof. In certain embodiments ofany of the aforementioned methods, the composition is applied to foliageof the soybean plant. In certain embodiments, the Methylobacterium sp.is selected for glyphosate resistance. In certain embodiments, thesoybean plant is a glyphosate tolerant soybean plant and a formulationcontaining glyphosate is also applied at about the V2 to about the V4stage of soybean plant development. In certain embodiments of any of theaforementioned methods, the methods can further comprise the step ofharvesting seed from the mature soybean plant. In certain embodiments,the yield of harvested seed is increased in comparison to yield ofharvested seed obtained from a control soybean plant that did notreceive an application of the Methylobacterium sp.

Also provided are soybean plants or soybean plant parts that are coatedor partially coated with a composition comprising a Methylobacterium sp.In certain embodiments, the soybean plant or soybean plant part iscoated or partially coated with a composition comprising aMethylobacterium sp. isolate selected from the group consisting of ISO02(NRRL B-50930), ISO03 (NRRL B-50931), ISO09 (NRRL B-50937), ISO10 (NRRLB-50938), and derivatives thereof. In certain embodiments, thecomposition comprises: (i) a solid substance with the Methylobacteriumgrown thereon and adhered thereto; or (ii) an emulsion having theMethylobacterium grown therein. In certain embodiments, the compositioncomprises the Methylobacterium sp. at a titer of about 1×10⁶ CFU/gm toabout 1×10¹⁴ CFU/gm for a solid composition or at a titer of about 1×10⁶CFU/mL to about 1×10¹¹ CFU/mL for a liquid composition containing thesolid substance or for the emulsion. In certain embodiments, theMethylobacterium sp. is Methylobacterium isolate is ISO09 (NRRLB-50937), ISO10 (NRRL B-50938), or a derivative thereof. In certainembodiments of any of the aforementioned compositions, the soybean plantpart is selected from the group consisting of a seed, a stem, a flower,a leaf, a petiole, a pod, and an axillary bud.

Also provided are methods for improving soybean plant yield thatcomprise: (i) applying a composition comprising a Methylobacterium sp.to a soybean seed or to a soybean plant at about the VE to about the V5stage of soybean plant development, wherein the composition comprises asolid substance with the Methylobacterium grown thereon and adheredthereto or an emulsion having the Methylobacterium grown therein; and,(ii) growing the soybean plant to maturity, thereby improving yield ofthe soybean plant. In certain embodiments, methods for improving soybeanplant yield that comprise: (a) applying a composition comprising aMethylobacterium sp. to a soybean seed or to a soybean plant at aboutthe VE to about the V5 stage of soybean plant development, wherein thecomposition comprises: (i) a solid substance with the Methylobacteriumgrown thereon and adhered thereto; (ii) an emulsion having theMethylobacterium grown therein; or (iii) a Methylobacterium isolateISO02 (NRRL B-50930), ISO03 (NRRL B-50931), ISO09 (NRRL B-50937), ISO10(NRRL B-50938), or a derivative thereof and an agriculturally acceptableadjuvant, excipient, or combination thereof; and (b) growing the soybeanplant to maturity, thereby improving yield of the soybean plant areprovided. In certain embodiments, the composition is applied at aboutthe V2 to about the V3 stage of development, about the V3 to about theV4 stage of development, or about the V3 stage of development. Incertain embodiments, the solid substance with the Methylobacterium grownthereon and adhered thereto is provided in a liquid or an emulsion. Incertain embodiments, the composition comprises the Methylobacterium sp.at a titer of about 1×10⁶ CFU/gm to about 1×10¹⁴ CFU/gm for a solidcomposition or at a titer of about 1×10⁶ CFU/mL to about 1×10¹¹ CFU/mLfor a liquid composition containing the solid substance or for theemulsion. In certain embodiments, the Methylobacterium sp. is ISO02(NRRL B-50930), ISO03 (NRRL B-50931), ISO09 (NRRL B-50937), ISO10 (NRRLB-50938), or a derivative thereof. In certain embodiments, theMethylobacterium sp. is Methylobacterium isolate ISO09 (NRRL B-50937),ISO10 (NRRL B-50938), or a derivative thereof. In certain embodiments,the composition comprises the solid substance or the emulsion and theMethylobacterium sp. is selected from the group consisting of ISO02(NRRL B-50930), ISO03 (NRRL B-50931), ISO09 (NRRL B-50937, ISO10 (NRRLB-50938), and derivatives thereof. In certain embodiments of any of theaforementioned methods, the derivative of the Methylobacterium isolateis selected for glyphosate resistance. In certain embodiments, thesoybean plant is a glyphosate tolerant soybean plant and a formulationcontaining glyphosate is also applied at about the V2 to about the V4stage of soybean plant development. In certain embodiments of any of theaforementioned methods, the method further comprises harvesting seedfrom the mature soybean plant. In certain embodiments of any of theaforementioned methods, the yield of harvested seed is increased incomparison to yield of harvested seed obtained from a control soybeanplant that did not receive an application of the Methylobacterium sp.

Also provided herein is an isolated Methylobacterium sp. strain ISO02(NRRL B-50930), ISO03 (NRRL B-50931), ISO09 (NRRL B-50937, ISO10 (NRRLB-50938), or derivative thereof. Compositions comprisingMethylobacterium sp. strain ISO02 (NRRL B-50930), ISO03 (NRRL B-50931),ISO09 (NRRL B-50937, ISO10 (NRRL B-50938), or derivative thereof arealso provided.

Also provided herein are methods for improving soybean plant early vigorthat comprise: (a) applying a composition comprising a Methylobacteriumsp. to a soybean seed or to a soybean plant at about the VE to about theV3 stage of soybean plant development, wherein the compositioncomprises: (i) a solid substance with the Methylobacterium grown thereonand adhered thereto; (ii) an emulsion having the Methylobacterium growntherein; or (iii) a Methylobacterium isolate ISO02 (NRRL B-50930), ISO03(NRRL B-50931), ISO09 (NRRL B-50937, ISO10 (NRRL B-50938), or aderivative thereof and an agriculturally acceptable adjuvant, excipient,or combination thereof; and, (b) growing a soybean plant from the seedor the soybean plant to the V3 to V6 stage of development, therebyimproving early vigor of the soybean plant. In certain embodiments ofthe methods, the composition is applied at about the VE to about the V2stage of development, about the VE to about the V1 stage of development,or VE stage of development. In certain embodiments of the methods, thecomposition comprises the Methylobacterium sp. at a titer of about 1×10⁶CFU/gm to about 1×10¹⁴ CFU/gm for a solid composition or at a titer ofabout 1×10⁶ CFU/mL to about 1×10¹¹ CFU/mL for a liquid compositioncontaining the solid substance or for the emulsion. In certainembodiments of the methods, the composition comprises the solid or theemulsion and the Methylobacterium sp. is Methylobacterium isolate ISO02(NRRL B-50930), ISO03 (NRRL B-50931), ISO09 (NRRL B-50937, ISO10 (NRRLB-50938), or a derivative thereof. In certain embodiments of themethods, the composition comprises the solid or the emulsion and theMethylobacterium sp. is Methylobacterium isolate ISO09 (NRRL B-50937,ISO10 (NRRL B-50938), or a derivative thereof. In certain embodiments ofthe methods, the derivative thereof is selected for resistance to abacteriocidal agent. In certain embodiments of the methods, theMethylobacterium isolate is selected for glyphosate resistance or forglufosinate resistance. In certain embodiments of the methods, thesoybean plant is a glyphosate tolerant soybean plant and a formulationcontaining glyphosate is also applied at about the V2 to about the V4stage of soybean plant development. In certain embodiments of any of theaforementioned methods, the vigor of the soybean plant in step (b) isincreased in comparison to vigor of a control soybean plant that did notreceive an application of the Methylobacterium sp. In certainembodiments of any of the aforementioned methods, increased vigorcomprises increased height, increased leaf area, increased chlorophyllcontent, increased stalk diameter, an advanced vegetative stage on aV1-V6 scale, root volume, root length, number of root tips, andcombinations thereof. In certain embodiments of the aforementionedmethods, the applied composition coats or partially coats the soybeanseed or the soybean plant or a part thereof.

DESCRIPTION Definitions

As used herein, the phrases “adhered thereto” and “adherent” refer toMethylobacterium that are associated with a solid substance by growing,or having been grown, on a solid substance.

As used herein, the phrase “agriculturally acceptable adjuvant” refersto a substance that enhances the performance of an active agent in acomposition for treatment of plants and/or plant parts. In certaincompositions, an active agent can comprise a mono-culture or co-cultureof Methylobacterium.

As used herein, the phrase “agriculturally acceptable excipient” refersto an essentially inert substance that can be used as a diluent and/orcarrier for an active agent in a composition for treatment of plantsand/or plant parts. In certain compositions, an active agent cancomprise a mono-culture or co-culture of Methylobacterium.

As used herein, the term “Methylobacterium” refers to bacteria that arefacultative methylotrophs of the genus Methylobacterium. The termMethylobacterium, as used herein, thus does not encompass includesspecies in the genera Methylobacter, Methylomonas, Methylomicrobium,Methylococcus, Methylosinus, Methylocystis, Methylosphaera,Methylocaldum, and Methylocella, which are obligate methanotrophs.

As used herein, the phrase “control plant” refers to a plant that hadnot received treatment with a yield or early vigor enhancingMethylobacterium or composition comprising the same at either the seedor any subsequent stage of the control plant's development. In certainembodiments, a control plant can be a plant that was treated with ayield neutral Methylobacterium sp.

As used herein, the phrase “co-culture of Methylobacterium” refers to aMethylobacterium culture comprising at least two strains ofMethylobacterium or at least two species of Methylobacterium.

As used herein, the phrase “contaminating microorganism” refers tomicroorganisms in a culture, fermentation broth, fermentation brothproduct, or composition that were not identified prior to introductioninto the culture, fermentation broth, fermentation broth product, orcomposition.

As used herein, the phrase “derivatives thereof”, when used in thecontext of a Methylobacterium isolate, refers to any strain that isobtained from the Methylobacterium isolate. Derivatives of aMethylobacterium isolate include, but are not limited to, variants ofthe strain obtained by selection, variants of the strain selected bymutagenesis and selection, and a genetically transformed strain obtainedfrom the Methylobacterium isolate.

As used herein, the phrase “early soybean vigor” or “early vigor”, whenused in the context of apply compositions containing Methylobacterium tosoybean seed, plants or parts of plants, refers to any growthcharacteristic of a soybean plant in the V3 to V6 stage of developmentthat is indicative of improved growth in comparison to an untreatedsoybean plant. Such growth characteristics can include, but are notlimited to, increased height, increased leaf area, increased chlorophyllcontent, increased stalk diameter, an advanced vegetative stage on aV1-V6 scale, increased root volume, increased root length, increasednumber of root tips, and combinations thereof.

As used herein, the term “emulsion” refers to a colloidal mixture of twoimmiscible liquids wherein one liquid is the continuous phase and theother liquid is the dispersed phase. In certain embodiments, thecontinuous phase is an aqueous liquid and the dispersed phase is liquidthat is not miscible, or partially miscible, in the aqueous liquid.

As used herein, the phrase “essentially free of contaminatingmicroorganisms” refers to a culture, fermentation broth, fermentationproduct, or composition where at least about 95% of the microorganismspresent by amount or type in the culture, fermentation broth,fermentation product, or composition are the desired Methylobacterium orother desired microorganisms of pre-determined identity.

As used herein, the phrase “inanimate solid substance” refers to asubstance which is insoluble or partially soluble in water or aqueoussolutions and which is either non-living or which is not a part of astill-living organism from which it was derived.

As used herein, the phrase “mono-culture of Methylobacterium” refers toa Methylobacterium culture consisting of a single strain ofMethylobacterium.

As used herein, the term “peptide” refers to any polypeptide of 50 aminoacid residues or less.

As used herein, the term “protein” refers to any polypeptide having 51or more amino acid residues.

As used herein, a “pesticide” refers to an agent that is insecticidal,fungicidal, nematocidal, bacteriocidal, or any combination thereof.

As used herein, the phrase “bacteriostatic agent” refers to agents thatinhibit growth of bacteria but do not kill the bacteria.

As used herein, the phrase “pesticide does not substantially inhibitgrowth of said Methylobacterium” refers to any pesticide that whenprovided in a composition comprising a fermentation product comprising asolid substance wherein a mono-culture or co-culture of Methylobacteriumis adhered thereto, results in no more than a 50% inhibition ofMethylobacterium growth when the composition is applied to a plant orplant part in comparison to a composition lacking the pesticide. Incertain embodiments, the pesticide results in no more than a 40%, 20%,10%, 5%, or 1% inhibition of Methylobacterium growth when thecomposition is applied to a plant or plant part in comparison to acomposition lacking the pesticide.

As used herein, the term “PPFM bacteria” refers without limitation tobacterial species in the genus Methylobacterium other than M. nodulans.

As used herein, the phrase “solid substance” refers to a substance whichis insoluble or partially soluble in water or aqueous solutions.

As used herein, the phrase “solid phase that can be suspended therein”refers to a solid substance that can be distributed throughout a liquidby agitation.

As used herein, the term “non-regenerable” refers to either a plant partor processed plant product that cannot be regenerated into a wholeplant.

As used herein, the phrase “substantially all of the solid phase issuspended in the liquid phase” refers to media wherein at least 95%,98%, or 99% of solid substance(s) comprising the solid phase aredistributed throughout the liquid by agitation.

As used herein, the phrase “substantially all of the solid phase is notsuspended in the liquid phase” refers to media where less than 5%, 2%,or 1% of the solid is in a particulate form that is distributedthroughout the media by agitation.

To the extent to which any of the preceding definitions is inconsistentwith definitions provided in any patent or non-patent referenceincorporated herein by reference, any patent or non-patent referencecited herein, or in any patent or non-patent reference found elsewhere,it is understood that the preceding definition will be used herein.

Yield or Early Vigor Enhancing Methylobacterium, Compositions ComprisingYield or Early Vigor Enhancing Methylobacterium, Methods of their Use,and Methods of Making

Various yield or early vigor enhancing Methylobacterium isolates,compositions comprising these Methylobacterium, methods of using thecompositions to improve soybean plant yield and/or early vigor, andmethods of making the compositions are provided herein. Amounts of thecompositions that comprise yield enhancing Methylobacterium sp.sufficient to provide for improved soybean plant yield can be determinedby measuring any or all of changes in yield relative to untreated plantsor plant parts. In certain embodiments, yield can be assessed bymeasuring output of seed on a per unit area basis (i.e. bushels peracre, kilograms per hectare, and the like), where the yield enhancingMethylobacterium sp treated plants or plants grown from Methylobacteriumsp treated seed are grown at about the same density as the controlplants. In certain embodiments, yield can be assessed by measuringoutput on a per plant or per plant part basis (grams of seed per plant,grams of seed per cob, kernels per plant, kernels per cob and the like)of the yield enhancing Methylobacterium sp treated plants in comparisonto untreated control plants.

Isolated yield or early vigor enhancing Methylobacterium sp. areprovided herein. In certain embodiments, the Methylobacterium isselected from the group consisting of M. aminovorans, M. extorquens, Mfujisawaense, M. mesophilicum, M. radiotolerans, M. rhodesianum, M.nodulans, M. phyllosphaerae, M. thiocyanatum, and M. oryzae. In certainembodiments, Methylobacterium is not M. radiotolerans or M. oryzae. Incertain embodiments, the yield or early vigor enhancing Methylobacteriumisolate is selected from the group consisting of ISO02, ISO03, ISO09,ISO10, and derivatives thereof. In certain embodiments, the yieldenhancing Methylobacterium isolate can enhance yield when applied priorto or during reproductive stages of soybean development and is aselected from the group consisting of ISO09, ISO10, and derivativesthereof. In certain embodiments, the yield or early vigor enhancingMethylobacterium isolate can enhance yield or early vigor when appliedto a soybean seed or in vegetative stages of soybean development and isselected from the group consisting of ISO02, ISO03, ISO10, andderivatives thereof. In certain embodiments, the yield enhancingMethylobacterium provides for at least about 2%, at least about 5%, atleast about 10%, or at least about 15% increases in yield of a treatedplant or a plant arising from a treated seed in comparison to untreatedcontrol plants or plants grown from untreated seeds. In certainembodiments, the yield enhancing Methylobacterium provides for at leastabout 2% or at least about 5% to at least about a 10% or at least abouta 20% increases in yield of a treated plant or a plant grown from atreated seed in comparison to untreated control plants or plants arisingfrom untreated seeds.

In certain embodiments, the Methylobacterium is not M. radiotolerans orM. oryzae. In certain embodiments, the yield or early vigor enhancingMethylobacterium provides for increases in yield or improved early vigorwhen applied to a seed. In certain embodiments, the yield enhancingMethylobacterium provides for increases in yield when applied just priorto or during soybean reproductive stages of development. In certainembodiments of any of the aforementioned compositions, the compositioncomprises a solid substance wherein a mono-culture or co-culture ofMethylobacterium is adhered thereto. In certain embodiments where theMethylobacterium is adhered to a solid substance, the compositioncomprises a colloid formed by the solid substance wherein a mono-cultureor co-culture of Methylobacterium is adhered thereto and a liquid. Incertain embodiments, the colloid is a gel. In certain embodiments ofcertain aforementioned compositions, composition is an emulsion thatdoes not contain a solid substance. In certain embodiments of any of theaforementioned compositions, the yield or early vigor enhancingMethylobacterium is selected from the group consisting of ISO02, ISO03,ISO09, ISO10, and derivatives thereof.

In certain embodiments, isolated yield or early vigor enhancingMethylobacterium sp. can be identified by treating a plant, a seed, soilin which the plant or a plant arising from the seed are grown, or otherplant growth media in which the plant or a plant arising from the seedare grown and assaying for increased yield or improved early vigor.

In certain embodiments, soybean seed or soybeans in the vegetativestages of development are treated with the yield or early vigorenhancing Methylobacterium sp. The vegetative stages of soybean are asfollows: VE (emergence), VC (cotyledon stage), V1 (first trifoliolateleaf), V2 (second trifoliolate leaf), V3 (third trifoliolate leaf), V4(fourth trifoliate leaf), up to V(n) (nth trifoliolate leaf, where thefinal number of trifoliate leaves depend on the soybean variety andenvironmental conditions). A description of the soybean vegetativestages can be found on the world wide web (internet) atextension.agron.iastate.edu/soybean/production_growthstages.html and in“Soybean Growth and Development”, Pedersen, P., Iowa State UniversityExtension and Outreach publication PM 1945, December 2009). In certainembodiments, the yield or early vigor enhancing Methylobacterium sp. areapplied at about the VE to about the V4, V5, V6, or Vn stage ofdevelopment, where n is the number of trifoliate leaves presentimmediately prior to entering the reproductive stages of development. Incertain embodiments, the yield enhancing Methylobacterium sp. areapplied at about the VC, V1, V2, or V3 to about the V4, V5, V6, or Vnstage of development, where n is the number of trifoliate leaves presentimmediately prior to entering the reproductive stages of development. Incertain embodiments, the yield or early vigor enhancing Methylobacteriumthat is applied to the seed or during the vegetative stage is selectedfrom the group consisting of ISO02, ISO03, ISO10, and derivativesthereof.

In certain embodiments, the yield or early vigor enhancingMethylobacterium are applied before, during, or after the application ofglyphosate to a transgenic soybean plant that is glyphosate tolerant.Commercially available glyphosate formulations that can be used include,but are not limited to, Roundup Original MAX®, Roundup PowerMAX®,Roundup UltraMax®, or RoundUp WeatherMAX® (Monsanto Co., St. Louis, Mo.,USA); Touchdown IQ® or Touchdown Total® (Syngenta, Wilmington, Del.,USA); Glyphomax®, Glyphomax Plus®, or Glyphomax XRT® (Dow AgrosciencesLLC, Indianapolis, Ind., USA). Soybean plants are typically sprayed withglyphosate at about the V3-V4 vegetative development stage. In certainembodiments, the yield or early vigor enhancing Methylobacterium that isapplied before, during, or after the application of glyphosate aMethylobacterium that is selected for glyphosate resistance. Selectionsfor glyphosate resistant bacteria that have been described (Comai etal., Science 221(4608):370-371) can be adapted for selection of yield orearly vigor enhancing Methylobacterium that are glyphosate resistant.The selection and use of glyphosate resistant yield or early vigorenhancing Methylobacterium from mutagenized or other populations ofMethylobacterium such as ISO02, ISO03, ISO09, and ISO10 is providedherein.

In certain embodiments, soybean seed or soybeans in the late vegetativestages to reproductive stages of development are treated with the yieldenhancing Methylobacterium sp. The late vegetative stages of soybean arethe V5 or V6, to V(n) (nth trifoliolate leaf, where the final number oftrifoliate leaves depend on the soybean variety and environmentalconditions) stages. The reproductive stages of soybean development are:R1 (beginning flowering-at least one flower on any node); R2 (fullflowering—an open flower at one of the two uppermost nodes); R3(beginning pod-pods are 5 mm at one of the four uppermost nodes); R4(full pod—pods at 2 cm at one of the four uppermost nodes); R5(Beginning seed-seed is 3 mm long in the pod at one of the fouruppermost nodes on the main stem); R6 (full seed-pod containing a greenseed that fills the pod capacity at one of the four uppermost nodes onthe main stem); R7 (beginning maturity-one normal pod on the main stemhas reached its mature pod color); and R8 (full maturity-95% of the podshave reached their full mature color. A description of the soybeanreproductive and vegetative stages can be found on the world wide web(internet) atextension.agron.iastate.edu/soybean/production_growthstages.html and in“Soybean Growth and Development”, Pedersen, P., Iowa State UniversityExtension and Outreach publication PM 1945, December 2009). In certainembodiments, the yield enhancing Methylobacterium sp. are applied atabout V5, V6, or Vn stage of development, where n is the number oftrifoliate leaves present immediately prior to entering the reproductivestages of development, to about the R2, R3, R4, R5, or R6 stage ofdevelopment. In certain embodiments, the yield enhancingMethylobacterium sp. are applied at about the V5, V6, or Vn stage ofdevelopment to about the R2, R3, or R4 stage of development. In certainembodiments, the yield enhancing Methylobacterium that is applied tolate vegetative or reproductive stage is selected from the groupconsisting of ISO09 and ISO10.

Various Methylobacterium sp. isolates provided herein are disclosed inTable 1.

TABLE 1 Methylobacterium sp. isolates USDA ARS ISOLATE No. NLS No. NRRLNo.¹ ISO01 NLS0046 NRRL B-50929 ISO02 NLS0020 NRRL B-50930 ISO03 NLS0017NRRL B-50931 ISO04 NLS0042 NRRL B-50932 ISO05 NLS0089 NRRL B-50933 ISO06NLS0068 NRRL B-50934 ISO07 NLS0065 NRRL B-50935 ISO08 NLS0069 NRRLB-50936 ISO09 NLS0062 NRRL B-50937 ISO10 NLS0064 NRRL B-50938 ISO11NLS0021 NRRL B-50939 ISO12 NLS0066 NRRL B-50940 ISO13 NLS0037 NRRLB-50941 ISO14 NLS0038 NRRL B-50942

¹ Deposit number for strain deposited with the AGRICULTURAL RESEARCHSERVICE CULTURE COLLECTION (NRRL) of the National Center forAgricultural Utilization Research, Agricultural Research Service, U.S.Department of Agriculture, 1815 North University Street, Peoria, Ill.61604 U.S.A. under the terms of the Budapest Treaty on the InternationalRecognition of the Deposit of Microorganisms for the Purposes of PatentProcedure. Subject to 37 CFR §1.808(b), all restrictions imposed by thedepositor on the availability to the public of the deposited materialwill be irrevocably removed upon the granting of any patent from thispatent application.

Co-assigned patent applications that disclose additional specific usesof the Methylobacterium strains of Table 1 such as: (1) increasing cornyield (U.S. 61/911,780, filed Dec. 4, 2013; and InternationalApplication claiming benefit of the same filed on Dec. 4, 2014); (2)improving lettuce cultivation (International Patent ApplicationPCT/US14/68558 filed on Dec. 4, 2014); and (3) improving tomato growth(International Patent Application PCT/US14/68611 filed on Dec. 4, 2014)and are each incorporated herein by reference in their entireties.Specifically incorporated herein by reference in their entireties arethe amino acid and genomic nucleic acid sequences of Methylobacteriumsp. NLS017, NLS020, NLS037, NLS042, NLS065, and NLS066 that aredisclosed in International Patent Application PCT/US14/68558 filed onDec. 4, 2014. Also specifically incorporated herein by reference intheir entireties are the amino acid and genomic nucleic acid sequencesof NLS017 and NLS066 disclosed in the International Patent ApplicationPCT/US14/68611, filed Dec. 4, 2014. Such amino acid and genomic nucleicacid sequences can be used to identify compositions, plant parts, plantseeds, or processed plant products provided herein that compriseMethylobacterium sp. NLS017 or NLS020.

Also provided herein are methods for improving soybean yield and/orearly vigor that comprise applying any of the aforementionedcompositions provided herein to a plant or a plant part in an amountthat provides for increased soybean yield in the plant, plant part, or aplant obtained therefrom relative to infection of a control plant, plantpart, or plant obtained therefrom that had not received an applicationof the composition. In certain embodiments, application of thecomposition provides for at least about 50%, at least about 75%, atleast about 85%, or at least about 95% increased soybean yield in theplant, plant part, or a plant derived therefrom relative to infection ofthe control plant, plant part, or plant obtained therefrom. In certainembodiments, the plant part is selected from the group consisting of aleaf, a stem, a flower, a root, a tuber, and a seed. In certainembodiments, the method further comprises the step of harvesting atleast one plant part selected from the group consisting of a leaf, astem, a flower, a root, a pod, or a seed from the plant or plant part.In certain embodiments of any of the aforementioned methods, the methodsfurther comprise obtaining a processed food or feed composition from theplant or plant part. In certain embodiments, the processed food or feedcomposition is a meal or a paste. In certain embodiments of any of theaforementioned methods, the yield or early vigor enhancingMethylobacterium is selected from the group consisting of ISO02, ISO03,ISO09, ISO10, and derivatives thereof. In certain embodiments where thecomposition is applied prior to or during the reproductive stages ofsoybean development, the yield enhancing Methylobacterium is ISO09,ISO10, or a derivative thereof. In certain embodiments where thecomposition is applied to the seed or during the vegetative stages ofsoybean development, the yield or early vigor enhancing Methylobacteriumis ISO02, ISO03, ISO10, or a derivative thereof.

Also provided are methods of making the compositions useful forimproving soybean yield that comprise combining a yield or early vigorenhancing Methylobacterium with an agriculturally acceptable excipientand/or with an agriculturally acceptable adjuvant. In certainembodiments of the methods, the Methylobacterium sp., is selected fromthe group consisting of M. aminovorans, M extorquens, M fujisawaense, M.mesophilicum, M. radiotolerans, M. rhodesianum, M nodulans, M.phyllosphaerae, M thiocyanatum, and M. oryzae. In certain embodiments ofthe methods, the Methylobacterium is not M. radiotolerans or M. oryzae.In certain embodiments of the methods, the Methylobacterium is adheredto a solid substance. In certain embodiments of the methods, theMethylobacterium is adhered to the solid substance is combined with aliquid to form a composition that is a colloid. In certain embodimentsof the methods, the colloid is a gel. In certain embodiments of themethods, the Methylobacterium adhered to the solid substance is providedby culturing the Methylobacterium in the presence of the solidsubstance. In certain embodiments of the methods, the compositioncomprises an emulsion. In certain embodiments of the methods, theMethylobacterium is provided by culturing the Methylobacterium in anemulsion. In certain embodiments of any of the aforementioned methods,the yield or early vigor enhancing Methylobacterium is selected from thegroup consisting of ISO02, ISO03, ISO09, and ISO10. In certainembodiments where the composition is applied prior to or during thereproductive stages of soybean development, the yield enhancingMethylobacterium is selected from the group consisting of ISO09 andISO10. In certain embodiments where the composition is applied to theseed or during the vegetative stages of soybean development, the yieldor early vigor enhancing Methylobacterium is ISO02, ISO03, and ISO10.

Methods where Methylobacterium are cultured in biphasic media comprisinga liquid phase and a solid substance have been found to significantlyincrease the resultant yield of Methylobacterium relative to methodswhere the Methylobacterium are cultured in liquid media alone. Incertain embodiments, the methods can comprise growing theMethylobacterium in liquid media with a particulate solid substance thatcan be suspended in the liquid by agitation under conditions thatprovide for Methylobacterium growth. In certain embodiments whereparticulate solid substances are used, at least substantially all of thesolid phase can thus be suspended in the liquid phase upon agitation.Such particulate solid substances can comprise materials that are about1 millimeter or less in length or diameter. In certain embodiments, thedegree of agitation is sufficient to provide for uniform distribution ofthe particulate solid substance in the liquid phase and/or optimallevels of culture aeration. However, in other embodiments providedherein, at least substantially all of the solid phase is not suspendedin the liquid phase, or portions of the solid phase are suspended in theliquid phase and portions of the solid phase are not suspended in theliquid phase. Non-particulate solid substances can be used in certainbiphasic media where the solid phase is not suspended in the liquidphase. Such non-particulate solid substances include, but are notlimited to, materials that are greater than about 1 millimeter in lengthor diameter. Such particulate and non-particulate solid substances alsoinclude, but are not limited to, materials that are porous, fibrous, orotherwise configured to provide for increased surface areas for adherentgrowth of the Methylobacterium. Biphasic media where portions of thesolid phase are suspended in the liquid phase and portions of the solidphase are not suspended in the liquid phase can comprise a mixture ofparticulate and non-particulate solid substances. Such particulate andnon-particulate solid substances used in any of the aforementionedbiphasic media also include, but are not limited to, materials that areporous, fibrous, or otherwise configured to provide for increasedsurface areas for adherent growth of the Methylobacterium. In certainembodiments, the media comprises a colloid formed by a solid and aliquid phase. A colloid comprising a solid and a liquid can bepre-formed and added to liquid media or can be formed in mediacontaining a solid and a liquid. Colloids comprising a solid and aliquid can be formed by subjecting certain solid substances to achemical and/or thermal change. In certain embodiments, the colloid is agel. In certain embodiments, the liquid phase of the media is anemulsion. In certain embodiments, the emulsion comprises an aqueousliquid and a liquid that is not miscible, or only partially miscible, inthe aqueous liquid. Liquids that are not miscible, or only partiallymiscible, in water include, but are not limited to, any of thefollowing: (1) liquids having a miscibility in water that is equal to orless than that of pentanol, hexanol, or heptanol at 25 degrees C.; (2)liquids comprising an alcohol, an aldehyde, a ketone, a fatty acid, aphospholipid, or any combination thereof; (3) alcohols selected from thegroup consisting of aliphatic alcohols containing at least 5 carbons andsterols; (4) an animal oil, microbial oil, synthetic oil, plant oil, orcombination thereof; and/or, (5) a plant oil is selected from the groupconsisting of soybean, soybean, cotton, peanut, sunflower, olive, flax,coconut, palm, rapeseed, sesame seed, safflower, and combinationsthereof. In certain embodiments, the immiscible or partially immiscibleliquid can comprises at least about 0.02% to about 20% of the liquidphase by mass. In certain embodiments, the methods can compriseobtaining a biphasic culture media comprising the liquid, the solid, andMethylobacterium and incubating the culture under conditions thatprovide for growth of the Methylobacterium. Biphasic culture mediascomprising the liquid, the solid, and Methylobacterium can be obtainedby a variety of methods that include, but are not limited to, any of (a)inoculating a biphasic media comprising the liquid and the solidsubstance with Methylobacterium; (b) inoculating the solid substancewith Methylobacterium and then introducing the solid substancecomprising the Methylobacterium into the liquid media; (c) inoculatingthe solid substance with Methylobacterium, incubating theMethylobacterium on the solid substance, and then introducing the solidsubstance comprising the Methylobacterium into the liquid media; or (d)any combination of (a), (b), or (c). Methods and compositions forgrowing Methylobacterium in biphasic media comprising a liquid and asolid are disclosed in co-assigned U.S. patent application Ser. No.13/907,161, filed May 31, 2013, which is incorporated herein byreference in its entirety, and in co-assigned International PatentApplication PCT/US13/43722, filed May 31, 2013, which is incorporatedherein by reference in its entirety.

Methods where Methylobacterium are cultured in media comprising anemulsion have also been found to significantly increase the resultantyield of Methylobacterium relative to methods where the Methylobacteriumare cultured in liquid media alone. In certain embodiments, the methodsfor making the compositions provided herein can comprise growing theyield or early vigor enhancing Methylobacterium agent in an emulsionunder conditions that provide for Methylobacterium growth. Mediascomprising the emulsion and yield or early vigor enhancingMethylobacterium can be obtained by a variety of methods that include,but are not limited to, any of: (a) inoculating a media comprising theemulsion with Methylobacterium; (b) inoculating the aqueous liquid withthe Methylobacterium, introducing the non-aqueous liquid, and mixing toform an emulsion; (c) inoculating the aqueous liquid with theMethylobacterium, introducing the non-aqueous liquid, and mixing to forman emulsion; or (d) any combination of (a), (b), or (c). In certainembodiments, the emulsion comprises an aqueous liquid and a liquid thatis not miscible, or only partially miscible, in the aqueous liquid.Non-aqueous liquids that are not miscible, or only partially miscible,in water include, but are not limited to, any of the following: (1)liquids having a miscibility in water that is equal to or less than thatof n-pentanol, n-hexanol, or n-heptanol at 25 degrees C.; (2) liquidscomprising an alcohol, an aldehyde, a ketone, a fatty acid, aphospholipid, or any combination thereof; (3) alcohols is selected fromthe group consisting of aliphatic alcohols containing at least 5, 6, or7 carbons and sterols; (4) an animal oil, microbial oil, synthetic oil,plant oil, or combination thereof; and/or, (5) a plant oil is selectedfrom the group consisting of soybean, soybean, cotton, peanut,sunflower, olive, flax, coconut, palm, rapeseed, sesame seed, safflower,and combinations thereof. In certain embodiments, the immiscible orpartially immiscible non-aqueous liquid can comprise at least about0.02% to about 20% of the emulsion by mass. In certain embodiments, theimmiscible or partially immiscible non-aqueous liquid can comprise atleast about any of about 0.05%, 0.1%, 0.5%, or 1% to about 3%, 5%, 10%,or 20% of the emulsion by mass. Methods and compositions for growingMethylobacterium in media comprising an emulsion are disclosed inco-assigned International Patent Application PCT/US2014/040218, filedMay 30, 2014, which is incorporated herein by reference in its entirety.

In certain embodiments, the fermentation broth, fermentation brothproduct, or compositions that comprise yield or early vigor enhancingMethylobacterium sp. can further comprise one or more introducedmicroorganisms of pre-determined identity other than Methylobacterium.Other microorganisms that can be added include, but are not limited to,microorganisms that are biopesticidal or provide some other benefit whenapplied to a plant or plant part. Biopesticidal or otherwise beneficialmicroorganisms thus include, but are not limited to, various Bacillussp., Pseudomonas sp., Coniothyrium sp., Pantoea sp., Streptomyces sp.,and Trichoderma sp. Microbial biopesticides can be a bacterium, fungus,virus, or protozoan. Particularly useful biopesticidal microorganismsinclude various Bacillus subtilis, Bacillus thuringiensis, Bacilluspumilis, Pseudomonas syringae, Trichoderma harzianum, Trichodermavixens, and Streptomyces lydicus strains. Other microorganisms that areadded can be genetically engineered or naturally occurring isolates thatare available as pure cultures. In certain embodiments, it isanticipated that the bacterial or fungal microorganism can be providedin the fermentation broth, fermentation broth product, or composition inthe form of a spore.

In certain embodiments, the liquid culture medium is prepared frominexpensive and readily available components, including, but not limitedto, inorganic salts such as potassium phosphate, magnesium sulfate andthe like, carbon sources such as glycerol, methanol, glutamic acid,aspartic acid, succinic acid and the like, and amino acid blends such aspeptone, tryptone, and the like. Exemplary liquid media that can be usedinclude, but are not limited to, ammonium mineral salts (AMS) medium(Whittenbury et al., 1970), Vogel-Bonner (VB) minimal culture medium(Vogel and Bonner, 1956), and LB broth (“Luria-Bertani Broth”).

In general, the solid substance used in the methods and compositionsthat provide for the efficient growth of Methylobacterium can be anysuitable solid substance which is insoluble or only partially soluble inwater or aqueous solutions. Such suitable solid substances are alsonon-bacteriocidal or non-bacteriostatic with respect to yield or earlyvigor enhancing Methylobacterium sp. when the solid substances areprovided in the liquid culture media. In certain embodiments, suchsuitable solid substances are also solid substances that are readilyobtained in sterile form or rendered sterile. Solid substances usedherein can be sterilized by any method that provides for removal ofcontaminating microorganisms and thus include, but are not limited to,methods such as autoclaving, irradiation, chemical treatment, and anycombination thereof. These solid substances include natural substancesof animal, plant, microbial, fungal, or mineral origin, manmadesubstances, or combinations of natural and manmade substances. Incertain embodiments, the solid substances are inanimate solidsubstances. Inanimate solid substances of animal, plant, microbial, orfungal origin can be obtained from animals, plants, microbes, or fungithat are inviable (i.e. no longer living) or that have been renderedinviable. Diatom shells are thus inanimate solid substances whenpreviously associated diatom algae have been removed or otherwiserendered inviable. Since diatom shells are inanimate solid substances,they are not considered to be photosynthetic organisms or photosyntheticmicroorganisms. In certain embodiments, solid substances include, butare not limited to, sand, silt, soil, clay, ash, charcoal, diatomaceousearth and other similar minerals, ground glass or glass beads, groundceramic materials, ceramic beads, bentonite, kaolin, talc, perlite,mica, vermiculite, silicas, quartz powder, montmorillonite, andcombinations thereof. In certain embodiments, the solid substance can bea polymer or polymeric beads. Polymers that can be used as a solidsubstance include, but are not limited to, various polysaccharides suchas cellulosic polymers and chitinous polymers which are insoluble oronly partially soluble in water or aqueous solutions, agar (i.e.galactans), and combinations thereof. In certain embodiments, the solidsubstance can be an insoluble or only partially soluble salt crystal.Salt crystals that can be used include, but are not limited to,insoluble or only partially soluble carbonates, chromates, sulfites,phosphates, hydroxides, oxides, and sulfides. In certain embodiments,the solid substance can be a microbial cell, fungal cell, microbialspore, or fungal spore. In certain embodiments, the solid substance canbe a microbial cell or microbial spore wherein the microbial cell ormicrobial spore is not a photosynthetic microorganism. In certainembodiments, the microbial cell or microbial spore is not aphotosynthetic microorganism, where the photosynthetic microorganism isselected from the group consisting of algae, cyanobacteria, diatoms,Botryococcus braunii, Chlorella, Dunaliella tertiolecta, Gracilaria,Pleurochrysis camerae, Sargassum, and Ulva. In still other embodiments,the solid substance can be an inactivated (i.e. inviable) microbialcell, fungal cell, microbial spore, or fungal spore. In still otherembodiments, the solid substance can be a quiescent (i.e. viable but notactively dividing) microbial cell, fungal cell, microbial spore, orfungal spore. In still other embodiments, the solid substance can becellular debris of microbial origin. In still other embodiments, thesolid substance can be particulate matter from any part of a plant.Plant parts that can be used to obtain the solid substance include, butare not limited to, cobs, husks, hulls, leaves, roots, flowers, stems,barks, seeds, and combinations thereof. Products obtained from processedplant parts including, but not limited to, bagasse, wheat bran, soygrits, crushed seed cake, stover, and the like can also be used. Suchplant parts, processed plants, and/or processed plant parts can bemilled to obtain the solid material in a particulate form that can beused. In certain embodiments, wood or a wood product including, but notlimited to, wood pulp, sawdust, shavings, and the like can be used. Incertain embodiments, the solid substance can be a particulate matterfrom an animal(s), including, but not limited to, bone meal, gelatin,ground or powdered shells, hair, macerated hide, and the like.

In certain embodiments, the solid substance is provided in a particulateform that provides for distribution of the solid substance in theculture media. In certain embodiments, the solid substance is comprisedof particle of about 2 microns to about 1000 microns in average lengthor average diameter. In certain embodiments, the solid substance iscomprised of particle of about 1 microns to about 1000 microns inaverage length or average diameter. In certain embodiments, the solidsubstance is a particle of about 1, 2, 4, 10, 20, or 40 microns to anyof about 100, 200, 500, 750, or 1000 microns in average length oraverage diameter. Desirable characteristics of particles used in themethods and compositions provided herein include suitable wettabilitysuch that the particles can be suspended throughout the media uponagitation.

In certain embodiments, the solid substance is provided in the media asa colloid wherein the continuous phase is a liquid and the dispersedphase is the solid. Suitable solids that can be used to form colloids inliquid media used to grow yield or early vigor enhancingMethylobacterium sp. include, but are not limited to, various solidsthat are referred to as hydrocolloids. Such hydrocolloids used in themedia, methods and compositions provided herein can be hydrophilicpolymers, of plant, animal, microbial, or synthetic origin. Hydrocolloidpolymers used in the methods can contain many hydroxyl groups and/or canbe polyelectrolytes. Hydrocolloid polymers used in the compositions andmethods provided herein include, but are not limited to, agar, alginate,arabinoxylan, carrageenan, carboxymethylcellulose, cellulose, curdlan,gelatin, gellan, β-glucan, guar gum, gum arabic, locust bean gum,pectin, starch, xanthan gum, and mixtures thereof. In certainembodiments, the colloid used in the media, methods, and compositionsprovided herein can comprise a hydrocolloid polymer and one or moreproteins.

In certain embodiments, the solid substance can be a solid substancethat provides for adherent growth of the yield or early vigor enhancingMethylobacterium sp. on the solid substance. Yield or early vigorenhancing Methylobacterium sp. that are adhered to a solid substance areMethylobacterium that cannot be substantially removed by simply washingthe solid substance with the adherent yield or early vigor enhancingMethylobacterium sp. with growth media whereas non-adherentMethylobacterium can be substantially removed by washing the solidsubstance with liquid growth media. In this context, “substantiallyremoved” means that at least about 30%, 40%, 50%, 60%, 70%, or 80% theMethylobacterium present are removed when the solid substance is washedwith three volumes of liquid growth media. Such washing can be effectedby a variety of methods including, but not limited to, decanting liquidfrom a washed solid phase or passing liquid through a solid phase on afilter that permits flow through of bacteria in the liquid. In certainembodiments, the adherent yield or early vigor enhancingMethylobacterium sp. that are associated with the solid can include bothMethylobacterium that are directly attached to the solid and/orMethylobacterium that are indirectly attached to the solid substance.Methylobacterium that are indirectly attached to the solid substanceinclude, but are not limited to, Methylobacterium that are attached toanother Methylobacterium or to another microorganism that is attached tothe solid substance, Methylobacterium that are attached to the solidsubstance by being attached to another substance that is attached to thesolid substance, and the like. In certain embodiments, at least 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5% or 99.9% ofthe Methylobacterium in the fermentation broth, fermentation brothproduct, or compositions are Methylobacterium that are adhered to thesolid substance. In certain embodiments, adherent yield or early vigorenhancing Methylobacterium sp. can be present on the surface of thesolid substance in the fermentation broth, fermentation broth product,or composition at a density of at least about 1 Methylobacterium/20square micrometers, of at least about 1 Methylobacterium/10 squaremicrometers, of at least about 1 Methylobacterium/10 square micrometers,of at least about 1 Methylobacterium/5 square micrometers, of at leastabout 1 Methylobacterium/2 square micrometers, or of at least about 1Methylobacterium/square micrometer. In certain embodiments, adherentyield or early vigor enhancing Methylobacterium sp. can be present onthe surface of the solid substance in the fermentation broth,fermentation broth product, or composition at a density of at leastabout 1 Methylobacterium/20 square micrometers to about 1Methylobacterium/square micrometer, of at least about 1Methylobacterium/10 square micrometers to about 1Methylobacterium/square micrometer, of at least about 1Methylobacterium/10 square micrometers to about 1Methylobacterium/square micrometer, of at least about 1Methylobacterium/5 square micrometers to about 1 Methylobacterium/squaremicrometer, or of at least about 1 Methylobacterium/2 square micrometersto about 1 Methylobacterium/square micrometer. In certain embodiments,adherent yield or early vigor enhancing Methylobacterium sp. can bepresent on the surface of the solid substance in the fermentation broth,fermentation broth product, or composition at a density of at leastabout 1 Methylobacterium/20 square micrometers to about 1Methylobacterium/2 square micrometers, of at least about 1Methylobacterium/10 square micrometers to about 1 Methylobacterium/2square micrometers, of at least about 1 Methylobacterium/10 squaremicrometers to about 1 Methylobacterium/2 square micrometers, or of atleast about 1 Methylobacterium/5 square micrometers to about 1Methylobacterium/2 square micrometers. Biphasic fermentation brothsprovided herein can comprise a liquid phase that contains non-adherentMethylobacterium. In certain embodiments, titers of non-adherentMethylobacterium in the liquid phase can be less than about 100,000,10,000, or 1,000 CFU/ml. In certain embodiments of any of theaforementioned compositions, the yield or early vigor enhancingMethylobacterium is selected from the group consisting of ISO02, ISO03,ISO09, ISO10, and derivatives thereof. In certain embodiments where thecomposition is applied prior to or during the reproductive stages ofsoybean development, the yield enhancing Methylobacterium is selectedfrom the group consisting of ISO09, ISO10, and derivatives thereof. Incertain embodiments where the composition is applied to the seed orduring the vegetative stages of soybean development, the yield or earlyvigor enhancing Methylobacterium is ISO02, ISO03, ISO10, or a derivativethereof.

Biphasic culture methods provided can yield fermentation broths withyield or early vigor enhancing Methylobacterium sp. at a titer ofgreater than about 5×10⁸ colony-forming units per milliliter, at a titerof greater than about 1×10⁹ colony-forming units per milliliter, at atiter of greater than about 1×10¹⁰ colony-forming units per milliliter,at a titer of at least about 3×10¹⁰ colony-forming units per milliliter.In certain embodiments, fermentation broths provided herein can compriseyield or early vigor enhancing Methylobacterium sp. at a titer of atleast about 5×10⁸ colony-forming units per milliliter to at least about3×10¹⁰ colony-forming units per milliliter, at least about 5×10⁸colony-forming units per milliliter to at least about 4×10¹⁰colony-forming units per milliliter, or at least about 5×10⁸colony-forming units per milliliter to at least about 6×10¹⁰colony-forming units per milliliter. In certain embodiments,fermentation broths provided herein can comprise yield or early vigorenhancing Methylobacterium sp. at a titer of at least about 1×10⁹colony-forming units per milliliter to at least about 3×10¹⁰colony-forming units per milliliter, at least about 1×10⁹ colony-formingunits per milliliter to at least about 4×10¹⁰ colony-forming units permilliliter, or at least about 1×10⁹ colony-forming units per milliliterto at least about 6×10¹⁰ colony-forming units per milliliter. In certainembodiments, fermentation broths provided herein will comprise yield orearly vigor enhancing Methylobacterium sp. at a titer of at least about1×10¹⁰ colony-forming units per milliliter to at least about 3×10¹⁰colony-forming units per milliliter, at least about 1×10¹⁰colony-forming units per milliliter to at least about 4×10¹⁰colony-forming units per milliliter, or at least about 1×10¹⁰colony-forming units per milliliter to at least about 6×10¹⁰colony-forming units per milliliter. In certain embodiments,fermentation broths provided herein will comprise yield or early vigorenhancing Methylobacterium sp. at a titer of, at least about 3×10¹⁰colony-forming units per milliliter to at least about 4×10¹⁰colony-forming units per milliliter, or at least about 3×10¹⁰colony-forming units per milliliter to at least about 6×10¹⁰colony-forming units per milliliter. In certain embodiments of any ofthe aforementioned compositions, the yield or early vigor enhancingMethylobacterium is selected from the group consisting of ISO02, ISO03,ISO09, ISO10, and derivatives thereof. In certain embodiments where thecomposition is applied prior to or during the reproductive stages ofsoybean development, the yield enhancing Methylobacterium is selectedfrom the group consisting of ISO09, ISO10, or a derivative thereof. Incertain embodiments where the composition is applied to the seed orduring the vegetative stages of soybean development, the yield or earlyvigor enhancing Methylobacterium is ISO02, ISO03, ISO10, and derivativesthereof.

Solid substances with adherent yield or early vigor enhancingMethylobacterium sp. can be obtained as fermentation products can beused to make various compositions useful for treating plants or plantparts to improve soybean yield. Alternatively, compositions providedherein comprising yield enhancing Methylobacterium sp., solid substanceswith yield enhancing Methylobacterium sp. grown thereon, or comprisingemulsions with yield enhancing Methylobacterium sp. grown therein can beused to treat plants or plant parts. Plants, plant parts, and, inparticular, plant seeds that have been at least partially coated orcoated with the fermentation broth products or compositions comprisingyield enhancing Methylobacterium sp. are thus provided. Partial coatingof a plant, a plant part, or a seed includes, but is not limited tocoating at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,95%, 98%, 99%, or about 99.5% of the surface area of the plant, plantpart, or plant seed. Also provided are processed plant products thatcontain the fermentation broth products or compositions with yield orearly vigor enhancing Methylobacterium sp. or adherent yield or earlyvigor enhancing Methylobacterium sp. Solid substances with adherentyield or early vigor enhancing Methylobacterium sp. can be used to makevarious compositions that are particularly useful for treating plantseeds. Seeds that have been at least partially coated with thefermentation broth products or compositions are thus provided. Partialcoating of a seed includes, but is not limited to coating at least about5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or about99.5% of the surface area of the seed. Also provided are processed seedproducts, including, but not limited to, meal, flour, feed, and flakesthat contain the fermentation broth products or compositions providedherein. In certain embodiments, the processed plant product will benon-regenerable (i.e. will be incapable of developing into a plant). Incertain embodiments, the solid substance used in the fermentationproduct or composition that at least partially coats the plant, plantpart, or plant seed or that is contained in the processed plant, plantpart, or seed product comprises a solid substance and associated oradherent yield or early vigor enhancing Methylobacterium sp. that can bereadily identified by comparing a treated and an untreated plant, plantpart, plant seed, or processed product thereof. In certain embodiments,the yield or early vigor enhancing Methylobacterium is selected from thegroup consisting of ISO02, ISO03, ISO09, ISO10, and derivatives thereof.

Compositions useful for treating plants or plant parts that compriseyield or early vigor enhancing Methylobacterium sp., a solid substancewith adherent yield or early vigor enhancing Methylobacterium sp., orcomprising emulsions with yield or early vigor enhancingMethylobacterium sp. grown therein can also further comprise anagriculturally acceptable adjuvant or an agriculturally acceptableexcipient. An agriculturally acceptable adjuvant or an agriculturallyacceptable excipient is typically an ingredient that does not causeundue phytotoxicity or other adverse effects when exposed to a plant orplant part. In certain embodiments, the solid substance can itself be anagriculturally acceptable adjuvant or an agriculturally acceptableexcipient so long as it is not bacteriocidal or bacteriostatic to theMethylobacterium. In other embodiments, the composition furthercomprises at least one of an agriculturally acceptable adjuvant or anagriculturally acceptable excipient. Any of the aforementionedcompositions can also further comprise a pesticide. Pesticides used inthe composition include, but are not limited to, an insecticide, afungicide, a nematocide, and a bacteriocide. In certain embodiments, thepesticide used in the composition is a pesticide that does notsubstantially inhibit growth of the Methylobacterium. AsMethylobacterium are gram negative bacteria, suitable bacteriocides usedin the compositions can include, but are not limited to, bacteriocidesthat exhibit activity against gram positive bacteria but not gramnegative bacteria. Compositions provided herein can also comprise abacteriostatic agent that does not substantially inhibit growth of theMethylobacterium. Bacteriostatic agents suitable for use in compositionsprovided herein include, but are not limited to, those that exhibitactivity against gram positive bacteria but not gram negative bacteria.Any of the aforementioned compositions can also be an essentially dryproduct (i.e. having about 5% or less water content), a mixture of thecomposition with an emulsion, or a suspension.

Agriculturally acceptable adjuvants used in the compositions thatcomprise yield or early vigor enhancing Methylobacterium sp. include,but are not limited to, components that enhance product efficacy and/orproducts that enhance ease of product application. Adjuvants thatenhance product efficacy can include various wetters/spreaders thatpromote adhesion to and spreading of the composition on plant parts,stickers that promote adhesion to the plant part, penetrants that canpromote contact of the active agent with interior tissues, extendersthat increase the half-life of the active agent by inhibitingenvironmental degradation, and humectants that increase the density ordrying time of sprayed compositions. Wetters/spreaders used in thecompositions can include, but are not limited to, non-ionic surfactants,anionic surfactants, cationic surfactants, amphoteric surfactants,organo-silicate surfactants, and/or acidified surfactants. Stickers usedin the compositions can include, but are not limited to, latex-basedsubstances, terpene/pinolene, and pyrrolidone-based substances.Penetrants can include mineral oil, vegetable oil, esterified vegetableoil, organo-silicate surfactants, and acidified surfactants. Extendersused in the compositions can include, but are not limited to, ammoniumsulphate, or menthene-based substances. Humectants used in thecompositions can include, but are not limited to, glycerol, propyleneglycol, and diethyl glycol. Adjuvants that improve ease of productapplication include, but are not limited to, acidifying/bufferingagents, anti-foaming/de-foaming agents, compatibility agents,drift-reducing agents, dyes, and water conditioners.Anti-foaming/de-foaming agents used in the compositions can include, butare not limited to, dimethopolysiloxane. Compatibility agents used inthe compositions can include, but are not limited to, ammonium sulphate.Drift-reducing agents used in the compositions can include, but are notlimited to, polyacrylamides, and polysaccharides. Water conditionersused in the compositions can include, but are not limited to, ammoniumsulphate.

Methods of treating plants and/or plant parts with the fermentationbroths, fermentation broth products, and compositions comprising yieldor early vigor enhancing Methylobacterium sp. are also provided herein.Treated plants, and treated plant parts obtained therefrom, include, butare not limited to, soybean. As used herein, the term soybean includes,but is not limited to all varieties, subspecies, and cultivars ofGlycine max. Soybean subspecies include, but are not limited to, Glycinemax L. ssp. max and Glycine max ssp. Formosana. Plant parts that aretreated include, but are not limited to, leaves, stems, flowers, roots,seeds, pods, and the like. Seeds or other propagules of any of theaforementioned plants can be treated with the fermentation broths,fermentation broth products, fermentation products, and/or compositionsprovided herein.

In certain embodiments, plants and/or plant parts are treated byapplying the fermentation broths, fermentation broth products,fermentation products, and compositions that comprise yield or earlyvigor enhancing Methylobacterium sp. as a spray. Such spray applicationsinclude, but are not limited to, treatments of a single plant part orany combination of plant parts. Spraying can be achieved with any devicethat will distribute the fermentation broths, fermentation brothproducts, fermentation products, and compositions to the plant and/orplant part(s). Useful spray devices include a boom sprayer, a hand orbackpack sprayer, crop dusters (i.e. aerial spraying), and the like.Spraying devices and or methods providing for application of thefermentation broths, fermentation broth products, fermentation products,and compositions to either one or both of the adaxial surface and/orabaxial surface can also be used. Plants and/or plant parts that are atleast partially coated with any of a biphasic fermentation broth, afermentation broth product, fermentation product, or compositions thatcomprise a solid substance with yield or early vigor enhancingMethylobacterium sp. adhered thereto are also provided herein. Partialcoating of a plant, a plant part, or a seed includes, but is not limitedto coating at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 95%, 98%, 99%, or about 99.5% of the surface area of the plant,plant part, or plant seed. Also provided herein are processed plantproducts that comprise a solid substance with yield or early vigorenhancing Methylobacterium sp. adhered thereto.

In certain embodiments, seeds are treated by exposing the seeds to thefermentation broths, fermentation broth products, fermentation products,and compositions that comprise yield or early vigor enhancingMethylobacterium sp. Seeds can be treated with the fermentation broths,fermentation broth products, and compositions provided herein by methodsincluding, but not limited to, imbibition, coating, spraying, and thelike. Seed treatments can be effected with both continuous and/or abatch seed treaters. In certain embodiments, the coated seeds can beprepared by slurrying seeds with a coating composition containing afermentation broth, fermentation broth product, or compositions thatcomprise the solid substance with yield or early vigor enhancingMethylobacterium sp. and air drying the resulting product. Air dryingcan be accomplished at any temperature that is not deleterious to theseed or the Methylobacterium, but will typically not be greater than 30degrees Centigrade. The proportion of coating that comprises a solidsubstance and yield or early vigor enhancing Methylobacterium sp.includes, but is not limited to, a range of 0.1 to 25% by weight of theseed, 0.5 to 5% by weight of the seed, and 0.5 to 2.5% by weight ofseed. In certain embodiments, a solid substance used in the seed coatingor treatment will have yield or early vigor enhancing Methylobacteriumsp. adhered thereon. In certain embodiments, a solid substance used inthe seed coating or treatment will be associated with yield or earlyvigor enhancing Methylobacterium sp. and will be a fermentation broth,fermentation broth product, or composition obtained by the methodsprovided herein. Various seed treatment compositions and methods forseed treatment disclosed in U.S. Pat. Nos. 5,106,648, 5,512,069, and8,181,388 are incorporated herein by reference in their entireties andcan be adapted for use with an active agent comprising the fermentationbroths, fermentation broth products, or compositions provided herein. Incertain embodiments, the composition used to treat the seed can containagriculturally acceptable excipients that include, but are not limitedto, woodflours, clays, activated carbon, diatomaceous earth, fine-graininorganic solids, calcium carbonate and the like. Clays and inorganicsolids that can be used with the fermentation broths, fermentation brothproducts, or compositions provided herein include, but are not limitedto, calcium bentonite, kaolin, china clay, talc, perlite, mica,vermiculite, silicas, quartz powder, montmorillonite and mixturesthereof. Agriculturally acceptable adjuvants that promote sticking tothe seed that can be used include, but are not limited to, polyvinylacetates, polyvinyl acetate copolymers, hydrolyzed polyvinyl acetates,polyvinylpyrrolidone-vinyl acetate copolymer, polyvinyl alcohols,polyvinyl alcohol copolymers, polyvinyl methyl ether, polyvinyl methylether-maleic anhydride copolymer, waxes, latex polymers, cellulosesincluding ethylcelluloses and methylcelluloses, hydroxymethylcelluloses, hydroxypropylcellulose, hydroxymethylpropylcelluloses,polyvinyl pyrrolidones, alginates, dextrins, malto-dextrins,polysaccharides, fats, oils, proteins, karaya gum, jaguar gum,tragacanth gum, polysaccharide gums, mucilage, gum arabics, shellacs,vinylidene chloride polymers and copolymers, soybean-based proteinpolymers and copolymers, lignosulfonates, acrylic copolymers, starches,polyvinylacrylates, zeins, gelatin, carboxymethylcellulose, chitosan,polyethylene oxide, acrylamide polymers and copolymers, polyhydroxyethylacrylate, methylacrylamide monomers, alginate, ethylcellulose,polychloroprene and syrups or mixtures thereof. Other usefulagriculturally acceptable adjuvants that can promote coating include,but are not limited to, polymers and copolymers of vinyl acetate,polyvinylpyrrolidone-vinyl acetate copolymer and water-soluble waxes.Various surfactants, dispersants, anticaking-agents, foam-controlagents, and dyes disclosed herein and in U.S. Pat. No. 8,181,388 can beadapted for use with an active agent comprising the fermentation broths,fermentation broth products, or compositions provided herein.

Provided herein are compositions that comprise yield or early vigorenhancing Methylobacterium sp. that provide for increase yield orimproved early vigor of soybean plants relative to untreated plants,plant parts, and plants obtained therefrom that have not been exposed tothe compositions. In certain embodiments, plant parts, including, butnot limited to, a seed, a leaf, a fruit, a stem, a root, a tuber, or acoleoptile can be treated with the compositions provided herein toincrease soybean plant yield. Treatments or applications can include,but are not limited to, spraying, coating, partially coating, immersing,and/or imbibing the plant or plant parts with the compositions providedherein. Partial coating of a plant, a plant part, or a seed includes,but is not limited to coating at least about 5%, 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or about 99.5% of the surfacearea of the plant, plant part, or plant seed. In certain embodiments, aseed, a leaf, a fruit, a stem, a root, a tuber, or a coleoptile can beimmersed and/or imbibed with a liquid, semi-liquid, emulsion, or slurryof a composition provided herein. Such seed immersion or imbibition canbe sufficient to provide for increased yield in a treated soybean plantor soybean plant grown from a treated seed in comparison to an untreatedsoybean plant or soybean plant grown from an untreated seed. In certainembodiments, plant seeds can be immersed and/or imbibed for at least 1,2, 3, 4, 5, or 6 hours. Such immersion and/or imbibition can, in certainembodiments, be conducted at temperatures that are not deleterious tothe plant seed or the Methylobacterium. In certain embodiments, theseeds can be treated at about 15 to about 30 degrees Centigrade or atabout 20 to about 25 degrees Centigrade. In certain embodiments, seedimbibition and/or immersion can be performed with gentle agitation. Incertain embodiments, the soybean seed or VE stage soybean plant isexposed to the composition by providing the composition in furrow.Providing the composition in furrow represents one of several methodsprovided herein for applying a composition to a soybean seed or to asoybean plant at about the VE stage of soybean plant development.

Compositions provided herein comprising yield or early vigor enhancingMethylobacterium sp. and related methods are therefore expected to beuseful in improving yield or early vigor in a wide variety of soybeanplants, including, but not limited to, all varieties, subspecies, andcultivars of Glycine max.

In certain embodiments, an amount of a composition provided herein thatis sufficient to provide for increased soybean yield or early vigor canbe a composition with yield or early vigor enhancing Methylobacteriumsp. at a titer of at least about 1×10⁶ colony-forming units permilliliter, at least about 5×10⁶ colony-forming units per milliliter, atleast about 1×10⁷ colony-forming units per milliliter, at least about5×10⁸ colony-forming units per milliliter, at least about 1×10⁹colony-forming units per milliliter, at least about 1×10¹⁰colony-forming units per milliliter, or at least about 3×10¹⁰colony-forming units per milliliter. In certain embodiments, an amountof a composition provided herein that is sufficient to provide forincreased soybean yield or early vigor to a plant or plant part can be acomposition with yield or early vigor enhancing Methylobacterium sp. ata titer of about least about 1×10⁶ colony-forming units per milliliter,at least about 5×10⁶ colony-forming units per milliliter, at least about1×10⁷ colony-forming units per milliliter, or at least about 5×10⁸colony-forming units per milliliter to at least about 6×10¹⁰colony-forming units per milliliter of a liquid or an emulsion. Incertain embodiments, an amount of a composition provided herein that issufficient to provide for increased soybean yield can be a fermentationbroth product with a yield or early vigor enhancing Methylobacterium sp.titer of a solid phase of that product is at least about 1×10⁶colony-forming units per milliliter, at least about 5×10⁶ colony-formingunits per milliliter, at least about 1×10⁷ colony-forming units permilliliter, or at least about 5×10⁸ colony-forming units per gram to atleast about 6×10¹⁰ colony-forming units of Methylobacterium per gram ofthe solid phase. In certain embodiments, an amount of a compositionprovided herein that is sufficient to provide for increased soybeanyield or early vigor can be a composition with a Methylobacterium titerof at least about 1×10⁶ colony-forming units per gram, at least about5×10⁶ colony-forming units per gram, at least about 1×10⁷ colony-formingunits per gram, or at least about 5×10⁸ colony-forming units per gram toat least about 6×10¹⁰ colony-forming units of Methylobacterium per gramof particles in the composition containing the particles that comprise asolid substance wherein a mono-culture or co-culture of yield or earlyvigor enhancing Methylobacterium sp. is adhered thereto. In certainembodiments, an amount of a composition provided herein that issufficient to provide for increased soybean yield to a plant or plantpart can be a composition with a Methylobacterium titer of at leastabout 1×10⁶ colony-forming units per mL, at least about 5×10⁶colony-forming units per mL, at least about 1×10⁷ colony-forming unitsper mL, or at least about 5×10⁸ colony-forming units per mL to at leastabout 6×10¹⁰ colony-forming units of Methylobacterium per mL in acomposition comprising an emulsion wherein a mono-culture or co-cultureof a yield or early vigor enhancing Methylobacterium sp. adhered to asolid substance is provided therein or grown therein. In certainembodiments, an amount of a composition provided herein that issufficient to provide for increased soybean yield to a plant or plantpart can be a composition with a Methylobacterium titer of at leastabout 1×10⁶ colony-forming units per mL, at least about 5×10⁶colony-forming units per mL, at least about 1×10⁷ colony-forming unitsper mL, or at least about 5×10⁸ colony-forming units per mL to at leastabout 6×10¹⁰ colony-forming units of Methylobacterium per mL of in acomposition comprising an emulsion wherein a mono-culture or co-cultureof a yield or early vigor enhancing Methylobacterium sp. is providedtherein or grown therein. In certain embodiments of any of theaforementioned compositions, the Methylobacterium sp. is selected fromthe group consisting of ISO02 (NRRL B-50930), ISO03 (NRRL B-50931),ISO09 (NRRL B-50937, ISO10 (NRRL B-50938), and derivatives thereof.

In certain embodiments, an amount of a composition provided herein thatis sufficient to provide for increased soybean yield can be acomposition with a Methylobacterium sp. at a titer of at least about1×10⁴ colony-forming units per milliliter, at least about 1×10⁵colony-forming units per milliliter, at least about 1×10⁶ colony-formingunits per milliliter, at least about 5×10⁶ colony-forming units permilliliter, at least about 1×10⁷ colony-forming units per milliliter, atleast about 5×10⁸ colony-forming units per milliliter, at least about1×10⁹ colony-forming units per milliliter, at least about 1×10¹⁰colony-forming units per milliliter, or at least about 3×10¹⁰colony-forming units per milliliter. In certain embodiments, an amountof a composition provided herein that is sufficient to provide forincreased soybean yield can be a composition with Methylobacterium sp.at a titer of at least about 1×10⁴ colony-forming units per milliliter,at least about 1×10⁵ colony-forming units per milliliter, about leastabout 1×10⁶ colony-forming units per milliliter, at least about 5×10⁶colony-forming units per milliliter, at least about 1×10⁷ colony-formingunits per milliliter, or at least about 5×10⁸ colony-forming units permilliliter to at least about 6×10¹⁰ colony-forming units per milliliterof a liquid or an emulsion. In certain embodiments, an amount of acomposition provided herein that is sufficient to provide for increasedsoybean yield can be a fermentation broth product with aMethylobacterium sp. titer of a solid phase of that product is at leastabout 1×10⁴ colony-forming units per gram, at least about 1×10⁵colony-forming units per gram, at least about 1×10⁶ colony-forming unitsper gram, at least about 5×10⁶ colony-forming units per gram, at leastabout 1×10⁷ colony-forming units per gram, or at least about 5×10⁸colony-forming units per gram to at least about 6×10¹⁰ colony-formingunits of Methylobacterium per gram, at least about 1×10¹¹ colony-formingunits of Methylobacterium per gram, at least about 1×10¹² colony-formingunits of Methylobacterium per gram, at least about 1×10¹³ colony-formingunits of Methylobacterium per gram, or at least about 5×10¹³colony-forming units of Methylobacterium per gram of the solid phase. Incertain embodiments, an amount of a composition provided herein that issufficient to provide for increased soybean yield can be a compositionwith a Methylobacterium titer of at least about 1×10⁶ colony-formingunits per gram, at least about 5×10⁶ colony-forming units per gram, atleast about 1×10⁷ colony-forming units per gram, or at least about 5×10⁸colony-forming units per gram to at least about 6×10¹⁰ colony-formingunits of Methylobacterium per gram, at least about 1×10¹¹ colony-formingunits of Methylobacterium per gram, at least about 1×10¹² colony-formingunits of Methylobacterium per gram, at least about 1×10¹³ colony-formingunits of Methylobacterium per gram, or at least about 5×10¹³colony-forming units of Methylobacterium per gram of particles in thecomposition containing the particles that comprise a solid substancewherein a mono-culture or co-culture of Methylobacterium sp. is adheredthereto. In certain embodiments, an amount of a composition providedherein that is sufficient to provide for increased soybean yield can bea composition with a Methylobacterium titer of at least about 1×10⁶colony-forming units per mL, at least about 5×10⁶ colony-forming unitsper mL, at least about 1×10⁷ colony-forming units per mL, or at leastabout 5×10⁸ colony-forming units per mL to at least about 6×10¹⁰colony-forming units of Methylobacterium per mL in a compositioncomprising an emulsion wherein a mono-culture or co-culture of aMethylobacterium sp. adhered to a solid substance is provided therein orgrown therein. In certain embodiments, an amount of a compositionprovided herein that is sufficient to provide for increased soybeanyield can be a composition with a Methylobacterium titer of at leastabout 1×10⁶ colony-forming units per mL, at least about 5×10⁶colony-forming units per mL, at least about 1×10⁷ colony-forming unitsper mL, or at least about 5×10⁸ colony-forming units per mL to at leastabout 6×10¹⁰ colony-forming units of Methylobacterium per mL of in acomposition comprising an emulsion wherein a mono-culture or co-cultureof a Methylobacterium sp. is provided therein or grown therein. Incertain embodiments of any of the aforementioned compositions, theMethylobacterium sp. is selected from the group consisting of ISO02(NRRL B-50930), ISO03 (NRRL B-50931), ISO09 (NRRL B-50937, ISO10 (NRRLB-50938), and derivatives thereof.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It will be appreciated by those of skill in the artthat the techniques disclosed in the following examples representtechniques discovered by the Applicants to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the instant disclosure, appreciate that many changes can bemade in the specific embodiments that are disclosed, while stillobtaining like or similar results, without departing from the scope ofthe invention.

Example 1 Increases in Soybean Yield by Application of MethylobacteriumCompositions

Soybean Foliar Field Trials

Soybean field trials were established at six Illinois, USA locations forthe purpose of evaluating 14 PPFM(pink-pigmented-facultative-methylotrophs of the speciesMethylobacterium) isolates applied as a foliar spray to soybean plantsat an early vegetative stage (V3) and also at a reproductive stage (R1).The locations were established at Cropsey, Dana, Homer, Farmer City,Farmington and Pesotum, all in Illinois.

Experimental Design

The trial was conducted as a split-plot design consisting of four30-inch rows and were 20 feet long. The two middle rows were thetreatment rows, the two outside rows were used as untreated border rows.There were eight replications of each of the 14 PPFM treatments forapplication at growth stages V3 and R1. The 14 PPFM treatments plus thecontrol (no PPFM, also referred to as “check”) comprised the whole plot,and the growth stage V3 and R1 comprised the split plot. There was a V3and R1 control included in each of the 8 replications.

Methods

In preparation for the field trials, the PPFM cultures were grown inAMS+glycerol+peptone+diatomaceous earth media at 30° C. for 6 days. Theammonium mineral salts (AMS) medium contains, per liter, 700 milligramsof dibasic potassium phosphate anhydrous, 540 milligrams of monobasicpotassium phosphate anhydrous, one gram of magnesium sulfateheptahydrate, 500 milligrams of ammonium chloride anhydrous, and 200milligrams of calcium chloride dihydrate.

AMS base medium was prepared from three stock solutions, listed below:

Stock solution I: for one liter at 50X concentration dibasic potassiumphosphate, anhydrous 35 grams monobasic potassium phosphate, anhydrous27 grams Stock solution II: for one liter at 50X concentration magnesiumsulfate heptahydrate 50 grams ammonium chloride, anhydrous 25 gramsStock solution III: for one liter at 50X concentration calcium chloridedihydrate 10 grams

Stock solutions I, II, and III were autoclaved separately.

To prepare one liter of liquid AMS medium with glycerol, peptone, anddiatomaceous earth, the following were added to 920 ml of distilledwater:

20 ml of stock solution I

20 ml of stock solution II

20 ml of stock solution III

20 ml of a 50% glycerol stock solution

10 grams of peptone

2 grams of diatomaceous earth

The resulting solution with suspended diatomaceous earth was sterilizedby autoclaving. The cultures were then harvested by centrifugation at5000 rpm for 15 minutes and then re-suspended in AMS+glycerol+peptonewith 20% glycerol as a cryoprotectant at 10× concentration. The cultureswere aliquoted and frozen at −80 until thawed for use. The liquid PPFMpreparations were applied to the soybean plants at the V3 or R1 stagesat a rate of 15 gal per acre using a backpack chemical sprayer. Titersof the PPFMs applied at the various locations for both the R1 and V3PPFM applications are provided in Tables 5 and 6, respectively. Thetrials were established within existing farmer field sites and weremanaged with local agronomic methods that the farmer practicesthroughout the growing season. All soybean varieties used were RoundupReady™ varieties, and the trials were sprayed with glyphosate at the V4stage of growth. The trials were harvested for yield at physiologicalmaturity with a commercial harvest combine. Table 2 indicates thevariety planted, planting date and harvest date at the soybean foliarsites.

TABLE 2 Variety planted, planting date and harvest date at the sixIllinois soybean foliar sites Location Cropsey, Farmer Farmington,Pesotum, IL Dana, IL City, IL IL Homer, IL IL Variety Great DuPont ™Monsanto ™ DuPont ™ DuPont ™ DuPont ™ Lakes ™ Pioneer ™ Asgrow ™Pioneer ™ Pioneer ™ Pioneer ™ GL3289R2 92Y75 3432 RJS38001 93Y84XBP37008 (Ovid, MI, (Johnston, (St. Louis, (Johnston, (Johnston,(Johnston, USA) IA, USA) MO) IA, USA) IA, USA) IA, USA) Planting dateMay 13, 2013 May 17, 2013 May 18, 2013 Jun. 13, 2013 May 18, 2013 May18, 2013 Harvest date Sep. 26, 2013 Sep. 23, 2013 Sep. 30, 2013 Oct. 16,2013 Oct. 11, 2013 Sep. 25, 2013

Results

Analysis of variance (ANOVA) was conducted with the Analyze-Fit Modelroutine in JMP version 11.0 (SAS Institute). After the parameterestimates were obtained from the models, plots of residuals and tablesof studentized residuals were examined for conformity with theassumptions of normality and constant variance. Comparisons of isolateswith the check within the same growth stage at application wereperformed with two-tailed t-tests applied to the pairwise differencesbetween least-squares means estimated from the ANOVA model, under thenull hypothesis that the difference in means was zero.

The following model was applied to the split plot design at the sixindividual locations:

Y _(ijk) =M+I _(i) +S _(j) +IS _(ij) +R _(k) +IR _(ik) +e _(ijk),  [1]

where Y_(ijk) is the yield of isolate i at stage j in replicate k, Mrepresents the overall mean, I_(i) is the fixed effect of isolate i,S_(j) is the fixed effect of stage j, IS_(ij) is the fixed effect of theinteraction of isolate i and stage j, R_(k) is the random effect ofreplicate k, IR_(ik) is the random effect of the interaction of isolatei and replicate k, and e_(ijk) is the random error.

Across-locations analyses for the six locations were conducted accordingto the following model:

Y _(hijk) =M+I _(i) +S _(j) +IS _(ij) +L _(h) +R(L)_(k(h)) +LI _(hi)+IR(L)_(ik(h)) +LS _(hj) +LIS _(hij) +e _(hijk),  [2]

where Y_(hijk) is the yield at location h of isolate i at stage j inreplicate k, M represents the overall mean, I_(i) is the fixed effect ofisolate i, S_(j) is the fixed effect of stage j, IS_(ij) is the fixedeffect of the interaction of isolate i and stage j, L_(h) is the randomeffect of location h, R(L)_(k(h)) is the random effect of replicate knested within location h, LI_(hi) is the random effect of theinteraction of location h and isolate i, IR(L)_(ik(h)) is the randomeffect of the interaction of isolate i and replicate k nested withinlocation h, LS_(hj) is the random effect of the interaction of locationh and stage j, LIS_(hij) is the random effect of the three-wayinteraction of location h with isolate i and stage j, and e_(hijk) isthe random error.

Seven of the 14 PPFM isolates showed a significant (p=0.25) yieldresponse vs the V3 or R1 check they were compared to in at least onelocation (Table 3). One isolate showed a significant increase vs thecheck across the six locations at both application stages (Table 4)

Five of 6 locations had at least one significant PPFM response.

TABLE 3 Mean yield, yield ranking, and p values of PPFM isolates at eachof six soybean foliar locations by growth stage Location Treatment StageYield Rank P Value Cropsey ISO01 V3 63.8 1 0.972 Cropsey Check V3 63.7 2— Cropsey ISO10 V3 63.2 3 0.833 Cropsey ISO14 V3 62.4 4 0.592 CropseyISO03 V3 61.9 5 0.442 Cropsey ISO07 V3 61.6 6 0.381 Cropsey ISO08 V361.5 7 0.358 Cropsey ISO05 V3 60.9 8 0.239 Cropsey ISO09 V3 60.7 9 0.219Cropsey ISO13 V3 60.6 10 0.193 Cropsey ISO06 V3 60.1 11 0.134 CropseyISO02 V3 60.0 12 0.131 Cropsey ISO04 V3 60.0 13 0.129 Cropsey ISO12 V359.5 14 0.085 Cropsey ISO11 V3 58.5 15 0.034 Cropsey ISO14 R1 63.5 10.568 Cropsey ISO01 R1 63.2 2 0.665 Cropsey Check R1 62.1 3 — CropseyISO10 R1 61.7 4 0.860 Cropsey ISO03 R1 61.6 5 0.817 Cropsey ISO07 R161.3 6 0.735 Cropsey ISO09 R1 61.1 7 0.675 Cropsey ISO08 R1 60.7 8 0.569Cropsey ISO11 R1 59.7 9 0.313 Cropsey ISO02 R1 59.7 10 0.310 CropseyISO06 R1 59.6 11 0.298 Cropsey ISO12 R1 59.6 12 0.290 Cropsey ISO05 R159.4 13 0.270 Cropsey ISO13 R1 58.7 14 0.154 Cropsey ISO04 R1 58.0 150.089 Dana ISO10 V3 56.5 1 0.092 Dana ISO03 V3 56.4 2 0.108 Dana ISO01V3 55.3 3 0.271 Dana ISO07 V3 55.3 4 0.271 Dana ISO09 V3 55.0 5 0.316Dana ISO02 V3 54.3 6 0.492 Dana ISO14 V3 54.3 7 0.508 Dana ISO12 V3 54.18 0.573 Dana ISO11 V3 54.1 9 0.574 Dana ISO08 V3 53.6 10 0.714 DanaCheck V3 52.8 11 — Dana ISO06 V3 52.8 12 0.993 Dana ISO05 V3 52.8 130.981 Dana ISO04 V3 51.6 14 0.573 Dana ISO13 V3 51.0 15 0.411 Dana ISO02R1 55.6 1 0.330 Dana ISO10 R1 55.1 2 0.451 Dana ISO12 R1 54.9 3 0.503Dana ISO06 R1 54.7 4 0.566 Dana ISO11 R1 54.7 5 0.575 Dana ISO09 R1 54.46 0.664 Dana ISO07 R1 54.2 7 0.732 Dana ISO03 R1 53.6 8 0.946 Dana ISO14R1 53.6 9 0.965 Dana Check R1 53.5 10 — Dana ISO01 R1 53.4 11 0.987 DanaISO08 R1 53.3 12 0.929 Dana ISO13 R1 52.7 13 0.723 Dana ISO05 R1 52.6 140.696 Dana ISO04 R1 51.1 15 0.284 F. City ISO07 V3 78.8 1 0.244 F. CityISO14 V3 78.4 2 0.299 F. City ISO04 V3 76.9 3 0.552 F. City ISO13 V376.7 4 0.606 F. City ISO10 V3 75.7 5 0.829 F. City Check V3 75.0 6 — F.City ISO08 V3 74.5 7 0.867 F. City ISO06 V3 74.4 8 0.859 F. City ISO02V3 74.1 9 0.766 F. City ISO12 V3 73.0 10 0.525 F. City ISO09 V3 72.7 110.473 F. City ISO05 V3 72.6 12 0.451 F. City ISO11 V3 72.0 13 0.347 F.City ISO01 V3 71.9 14 0.334 F. City ISO03 V3 70.4 15 0.152 F. City ISO07R1 76.8 1 0.773 F. City ISO13 R1 76.6 2 0.828 F. City ISO10 R1 76.2 30.916 F. City Check R1 75.9 4 — F. City ISO14 R1 75.0 5 0.795 F. CityISO02 R1 74.8 6 0.727 F. City ISO05 R1 73.8 7 0.526 F. City ISO06 R173.6 8 0.474 F. City ISO04 R1 73.3 9 0.429 F. City ISO08 R1 73.2 100.411 F. City ISO09 R1 73.0 11 0.364 F. City ISO12 R1 71.8 12 0.202 F.City ISO11 R1 71.8 13 0.200 F. City ISO03 R1 71.4 14 0.162 F. City ISO01R1 71.4 15 0.159 Farmington ISO10 V3 60.8 1 0.11  Farmington ISO14 V359.0 2 0.40  Farmington ISO12 V3 58.8 3 0.45  Farmington ISO09 V3 58.7 40.47  Farmington ISO07 V3 58.4 5 0.56  Farmington ISO03 V3 58.1 6 0.64 Farmington ISO13 V3 58.0 7 0.69  Farmington ISO02 V3 57.9 8 0.71 Farmington ISO01 V3 57.7 9 0.77  Farmington ISO06 V3 57.4 10 0.88 Farmington Check V3 57.0 11 — Farmington ISO04 V3 57.0 12 0.99 Farmington ISO05 V3 56.7 13 0.90  Farmington ISO08 V3 56.7 14 0.87 Farmington ISO11 V3 55.5 15 0.52  Farmington ISO10 R1 60.7 1 0.05 Farmington ISO13 R1 60.3 2 0.08  Farmington ISO12 R1 59.2 3 0.19 Farmington ISO09 R1 59.0 4 0.22  Farmington ISO03 R1 58.7 5 0.27 Farmington ISO07 R1 58.3 6 0.36  Farmington ISO14 R1 58.1 7 0.40 Farmington ISO06 R1 57.9 8 0.46  Farmington ISO05 R1 57.0 9 0.72 Farmington ISO08 R1 56.9 10 0.75  Farmington ISO02 R1 56.8 11 0.77 Farmington Check R1 56.1 12 — Farmington ISO01 R1 55.8 13 0.88 Farmington ISO11 R1 55.7 14 0.86  Farmington ISO04 R1 49.0 15 0.88 Homer ISO10 V3 83.2 1 0.34  Homer ISO09 V3 81.9 2 0.58  Homer ISO12 V380.9 3 0.81  Homer ISO03 V3 80.4 4 0.96  Homer Check V3 80.2 5 — HomerISO14 V3 79.2 6 0.74  Homer ISO04 V3 78.9 7 0.67  Homer ISO13 V3 78.6 80.59  Homer ISO06 V3 78.5 9 0.59  Homer ISO08 V3 78.5 10 0.58  HomerISO05 V3 78.4 11 0.56  Homer ISO11 V3 77.1 12 0.31  Homer ISO02 V3 76.913 0.29  Homer ISO01 V3 75.5 14 0.13  Homer ISO07 V3 74.9 15 0.08  HomerISO09 R1 81.5 1 0.21  Homer ISO10 R1 81.2 2 0.18  Homer ISO12 R1 80.9 30.22  Homer ISO11 R1 79.2 4 0.50  Homer ISO14 R1 79.2 5 0.50  HomerISO13 R1 78.8 6 0.57  Homer ISO07 R1 78.7 7 0.60  Homer ISO05 R1 78.6 80.62  Homer ISO02 R1 78.6 9 0.62  Homer ISO03 R1 78.5 10 0.63  HomerISO01 R1 78.2 11 0.72  Homer ISO08 R1 77.9 12 0.78  Homer ISO04 R1 77.713 0.83  Homer Check R1 77.1 14 — Homer ISO06 R1 77.1 15 0.99  PesotumISO02 V3 63.9 1 0.13  Pesotum ISO08 V3 62.4 2 0.42  Pesotum ISO13 V362.4 3 0.43  Pesotum ISO01 V3 61.9 4 0.55  Pesotum ISO14 V3 61.8 5 0.59 Pesotum ISO09 V3 61.1 6 0.84  Pesotum ISO05 V3 60.9 7 0.90  PesotumISO06 V3 60.9 8 0.91  Pesotum ISO10 V3 60.8 9 0.94  Pesotum ISO07 V360.8 10 0.94  Pesotum Check V3 60.6 11 — Pesotum ISO12 V3 60.6 12 1.00 Pesotum ISO03 V3 60.0 13 0.76  Pesotum ISO04 V3 59.7 14 0.67  PesotumISO11 V3 59.5 15 0.62  Pesotum ISO02 R1 63.0 1 0.32  Pesotum ISO03 R162.8 2 0.36  Pesotum ISO07 R1 62.4 3 0.46  Pesotum ISO08 R1 61.6 4 0.73 Pesotum ISO09 R1 61.2 5 0.86  Pesotum ISO04 R1 61.2 6 0.87  PesotumISO01 R1 61.1 7 0.89  Pesotum ISO13 R1 60.9 8 0.94  Pesotum Check R160.8 9 — Pesotum ISO14 R1 60.4 10 0.87  Pesotum ISO05 R1 60.2 11 0.79 Pesotum ISO11 R1 60.1 12 0.76  Pesotum ISO10 R1 60.0 13 0.73  PesotumISO12 R1 59.6 14 0.60  Pesotum ISO06 R1 58.2 15 0.24 

TABLE 4 Mean yield, yield ranking, and p values of PPFM isolates acrosscombined six combined soybean foliar locations by growth stage PPFMYield Location Treatment Stage bu/acre Rank P value Across 6 LocationsISO10 V3 67.9 1 0.09 Across 6 Locations ISO14 V3 67.2 2 0.37 Across 6Locations Check V3 66.5 3 — Across 6 Locations ISO09 V3 66.3 4 0.90Across 6 Locations ISO07 V3 66.3 5 0.97 Across 6 Locations ISO08 V3 66.16 0.71 Across 6 Locations ISO02 V3 65.9 7 0.72 Across 6 Locations ISO13V3 65.8 8 0.71 Across 6 Locations ISO03 V3 65.8 9 0.71 Across 6Locations ISO01 V3 65.7 10 0.60 Across 6 Locations ISO12 V3 65.6 11 0.69Across 6 Locations ISO04 V3 65.4 12 0.40 Across 6 Locations ISO06 V365.3 13 0.40 Across 6 Locations ISO05 V3 65.1 14 0.26 Across 6 LocationsISO11 V3 64.2 15 0.04 Across 6 Locations ISO10 R1 66.9 1 0.13 Across 6Locations ISO07 R1 66.7 2 0.32 Across 6 Locations ISO14 R1 66.3 3 0.49Across 6 Locations ISO02 R1 66.3 4 0.64 Across 6 Locations Check R1 65.95 — Across 6 Locations ISO09 R1 65.8 6 0.51 Across 6 Locations ISO03 R165.6 7 0.86 Across 6 Locations ISO13 R1 65.5 8 0.69 Across 6 LocationsISO01 R1 65.4 9 0.70 Across 6 Locations ISO12 R1 65.4 10 0.93 Across 6Locations ISO08 R1 65.3 11 0.77 Across 6 Locations ISO11 R1 65.1 12 0.49Across 6 Locations ISO05 R1 64.9 13 0.55 Across 6 Locations ISO06 R164.6 14 0.48 Across 6 Locations ISO04 R1 64.3 15 0.23

TABLE 5 Titers of PPFMs Applied at the R1 stage at Various Locations (inCFU/mL) Cropsey Pesotum Farmer City Homer Dana Farmington NLS # IsolateTiter Titer Titer Titer Titer Titer 0046 ISO01 3.2E+08 8.6E+08 8.6E+085.6E+08 8.6E+08 5.6E+08 0020 ISO02 1.0E+09 1.2E+09 7.4E+08 1.2E+091.2E+09 1.2E+09 0017 ISO03 4.6E+08 7.4E+08 2.8E+08 5.7E+08 2.8E+085.7E+08 0042 ISO04 3.9E+08 2.4E+08 2.4E+08 1.4E+08 2.4E+08 1.4E+08 0089ISO05 1.7E+08 1.7E+08 4.8E+08 6.7E+08 6.7E+08 6.7E+08 0068 ISO06 5.0E+082.4E+08 2.6E+08 2.6E+08 2.6E+08 2.4E+08 0065 ISO07 3.7E+08 3.8E+083.8E+08 4.2E+08 3.8E+08 3.7E+08 0069 ISO08 2.4E+08 2.0E+08 2.0E+082.7E+08 5.7E+08 2.7E+08 0062 ISO09 1.5E+08 1.0E+08 1.0E+08 3.4E+081.0E+08 2.9E+08 0064 ISO10 9.5E+08 5.9E+08 8.4E+08 9.0E+08 9.0E+083.1E+08 0021 ISO11 ND¹ 9.7E+08 9.7E+07 1.2E+08 9.7E+07 1.2E+08 0066ISO12 2.9E+08 5.6E+08 5.6E+08 3.4E+08 5.6E+08 4.8E+08 0037 ISO13 2.3E+082.3E+08 2.3E+08 ND¹ 2.3E+08 ND¹ 0038 ISO14 2.6E+07 1.6E+08 5.0E+076.7E+08 6.7E+07 1.3E+08 ¹ND: Not determined.

TABLE 6 Titers of PPFMs Applied at the V3 stage at Various Locations (inCFU/mL) Cropsey Pesotum Farmer City Homer Dana Farmington NLS # IsolateTiter Titer Titer Titer Titer Titer 0046 ISO01 4.2E+08 5.3E+08 3.2E+085.3E+08 5.3E+08 5.6E+08 0020 ISO02 1.0E+09 1.1E+09 1.1E+09 1.1E+096.0E+08 9.5E+08 0017 ISO03 6.2E+08 5.5E+08 5.9E+08 4.1E+08 4.6E+085.7E+08 0042 ISO04 2.2E+08 2.3E+08 2.2E+08 3.9E+08 2.3E+08 1.4E+08 0089ISO05 1.2E+08 1.2E+08 1.2E+08 1.7E+08 1.2E+08 6.7E+08 0068 ISO06 4.3E+082.9E+08 2.9E+08 6.2E+08 ND¹ 3.1E+08 0065 ISO07 2.9E+08 2.9E+08 2.9E+083.7E+08 3.7E+08 4.2E+08 0069 ISO08 1.9E+08 1.9E+08 2.0E+08 3.4E+081.9E+08 2.7E+08 0062 ISO09 1.5E+08 1.5E+08 1.5E+08 1.5E+08 1.1E+083.4E+08 0064 ISO10 9.5E+08 9.5E+08 9.5E+08 9.5E+08 9.5E+08 8.4E+08 0021ISO11 ND¹ ND¹ ND¹ ND¹ ND¹ 1.2E+08 0066 ISO12 3.0E+08 2.7E+08 3.0E+082.9E+08 2.9E+08 3.4E+08 0037 ISO13 ND¹ ND¹ ND¹ 2.3E+08 2.3E+08 ND¹ 0038ISO14 2.6E+07 2.6E+07 2.6E+07 2.6E+07 2.6E+07 1.6E+08 ¹ND: Notdetermined.

Example 2 Results of 2014 Field Tests

Strains for use in 2014 field trials were selected based on resultsfrom 1) previous field trials conducted in the summer of 2013 and 2)laboratory and controlled environment assays. All three strains used in2014 soy field trials demonstrated a positive effect on soy yield in the2013 field trials and beneficial effects on various agronomic traits inlaboratory and controlled environment assays.

In spring of 2014, field trials designed to evaluate the effect of thethree selected PPFM strains on soybean yield were established at sitesthroughout the Midwest soybean growing area. These included sites inIowa, Illinois, Nebraska, Missouri, Ohio, and Wisconsin. At each site,the experiment was set-up as a Randomized Complete Block Design with sixreplications. The minimum plot sizes were four rows of 20′ each. Allobservations were taken from the center two rows of each plot anddestructive samples were collected only from plot border rows. Rowspacing and plot maintenance were in accordance with standard growerpractices at each site.

A total of 13 treatments were applied at each site (Table 7). Each PPFMstrain was applied in two different manners (in-furrow and foliar) at ahigh and low rate, which differed between the two application methods.In-furrow treatments were applied at the time of planting by sprayapplication over the open seed furrow. Foliar applications were appliedat the V3 stage using a conventional boom. All applications were madewith either flat fan or cone jet nozzles.

Early plant vigor was rated 20 and 60 days after emergence. Vigorratings were given on a 1-5 scale based on visual assessment, with 1being poor and 5 being excellent. Various parameters, including plantheight, leaf area, color, and percent canopy closure, and incorporatedinto the visual assessment of plant vigor. Harvest was done per standardgrower practices and plot yields were determined using conventionalmetered combine harvesters. Plot test weights were used to calculatebu/acre yield estimates for each plot.

TABLE 7 Treatments used in 2014 field trials Appli- Num- cation Rate¹Titer range² ber Name Treatment Timing (mL/acre) (CFU/mL) 1 Check CheckUntreated 2 17_L_Inf NLS0017 In-furrow 625 4.7 × 10⁸-2.2 × 10⁹ 317_H_Inf NLS0017 In-furrow 1,250 4.7 × 10⁸-2.2 × 10⁹ 4 17_L_Fol NLS0017Foliar 2,500 4.7 × 10⁸-2.2 × 10⁹ 5 17_H_Fol NLS0017 Foliar 5,000 4.7 ×10⁸-2.2 × 10⁹ 6 20_L_Inf NLS0020 In-furrow 625 3.0 × 10⁸-3.1 × 10⁹ 720_H_Inf NLS0020 In-furrow 1,250 3.0 × 10⁸-3.1 × 10⁹ 8 20_L_Fol NLS0020Foliar 2,500 3.0 × 10⁸-3.1 × 10⁹ 9 20_H_Fol NLS0020 Foliar 5,000 3.0 ×10⁸-3.1 × 10⁹ 10 64_L_Inf NLS0064 In-furrow 625 3.7 × 10⁷-5.4 × 10⁸ 1164_H_Inf NLS0064 In-furrow 1,250 3.7 × 10⁷-5.4 × 10⁸ 12 64_L_Fol NLS0064Foliar 2,500 3.7 × 10⁷-5.4 × 10⁸ 13 64_H_Fol NLS0064 Foliar 5,000 3.7 ×10⁷-5.4 × 10⁸ ¹‘Rate’ indicates the number of mL concentrated productapplied per acre; this amount was diluted in a minimum of 5 gal/acretotal volume for in-furrow applications and 15 gal/acre for foliarapplications. ²‘Titer range’ provides the lowest and highest titers forPPFM concentrates at the time of shipping to trial sites.

Results

Yield and early vigor data were analyzed using the JMP statisticalanalysis software package (Version 9.0). The full model with all randomeffects was fit first and then reduced to the best fitting model basedon Akaike information criterion (AIC) values. Across locations meanscomparisons were conducted using Fisher's protected LSD test withα=0.05, 0.10, and 0.20 (Table 8; Table 9).

TABLE 8 Yield increase across locations Mean yield Yield > Check Yield >Check Yield > Check Number Treatment (bu/acre) at α = 0.05 at α = 0.10at α = 0.20 Rank 1 Check 62.20 12 2 17_L_Inf 63.39 9 3 17_H_Inf 65.17 XX X 1 4 17_L_Fol 62.00 13 5 17_H_Fol 65.09 X X 2 6 20_L_Inf 63.17 10 720_H_Inf 64.63 X 4 8 20_L_Fol 63.78 6 9 20_H_Fol 63.75 7 10 64_L_Inf62.70 11 11 64_H_Inf 64.15 X 5 12 64_L_Fol 63.55 8 13 64_H_Fol 65.00 X X3

TABLE 9 Vigor (20 days post-emergence) increase across locations MeanVigor > Check Vigor > Check Vigor > Check Number Treatment vigor at α =0.05 at α = 0.10 at α = 0.20 Rank 1 Check 3.60 9 2 17_L_Inf 3.62 7 317_H_Inf 3.89 X X 1 4 17_L_Fol 3.58 11 5 17_H_Fol 3.64 5 6 20_L_Inf 3.646 7 20_H_Inf 3.69 4 8 20_L_Fol 3.61 8 9 20_H_Fol 3.44 13 10 64_L_Inf3.75 2 11 64_H_Inf 3.72 3 12 64_L_Fol 3.49 12 13 64_H_Fol 3.60 10

Treatment yield relative to the check for individual locations wasanalyzed in the same manner as the across locations data (Table 4). Inthe table below, yields significantly greater than the check at α=0.05,0.10, and 0.20 are represented by ‘XXX,’ XX,′ and ‘X,’ respectively.

TABLE 10 Yield increase over check by location Number Treatment AgProBuckeye FC Homer Maloney Rains RFR 1 Check 2 17_L_Inf XXX 3 17_H_Inf XXXX XX 4 17_L_Fol XXX 5 17_H_Fol XXX XX 6 20_L_Inf XXX X 7 20_H_Inf XXXXXX 8 20_L_Fol XXX 9 20_H_Fol XXX XXX 10 64_L_Inf XXX XXX 11 64_H_InfXXX XXX 12 64_L_Fol XXX 13 64_H_Fol XXX XXX XXX

At many locations, PPFM-treated plots had greater yields than the checkbut not at a statistically significant level. Due to the inherentvariability of field research, these yield increases could stillindicate significant biological effects by PPFMs. The yield increasesfacilitated by PPFM-application could also result in significantfinancial gains across large acreages. Consequently, yield values foreach site are reported below and treatment yields numerically greaterthan the check have been highlighted (bolded and underlined) todemonstrate positive trends across locations (Table 11).

TABLE 11 Average yields (bu/acre) by location Number Treatment AgProBuckeye FC Homer Maloney Rains RFR 1 Check 45.11 58.94 74.11 61.56 68.4345.50 81.76 2 17_L_Inf 52.65 56.65 71.16 64.74 69.49 48.10 80.94 317_H_Inf 52.65 59.46 72.10 69.21 72.63 49.40 80.75 4 17_L_Fol 51.2559.04 67.52 61.17 69.86 48.10 77.01 5 17_H_Fol 57.62 60.32 75.15 64.8173.20 48.53 75.95 6 20_L_Inf 55.20 58.56 72.76 59.24 70.09 50.27 76.09 720_H_Inf 61.17 57.02 70.64 62.34 75.58 47.67 78.00 8 20_L_Fol 53.0055.79 75.85 63.99 70.25 49.40 78.17 9 20_H_Fol 52.66 58.20 75.44 61.4876.06 48.53 73.54 10 64_L_Inf 52.17 57.00 67.61 63.84 74.42 45.50 78.3111 64_H_Inf 52.83 59.11 74.05 60.38 73.15 48.97 80.53 12 64_L_Fol 57.4957.54 77.99 58.15 69.18 46.37 78.10 13 64_H_Fol 58.59 56.97 72.07 65.9474.41 52.87 74.11

CONCLUSIONS

Three elite PPFM isolates were evaluated for their ability to increasesoybean yield in 2014 field trials. In these trials, all treatmentssignificantly increased yield over a check at a minimum of one location.Across locations, the high rates of all isolates applied as either anin-furrow or a foliar treatment, with the exception of NLS0020in-furrow, significantly increased soy yields at α=0.20. The high rateof both NLS0017 foliar and in-furrow treatments and NLS0064 foliarincreased yield over the check at all locations when a more stringentlevel of a was applied. The high rate of NLS0017 in-furrow was the onlytreatment to significantly increase vigor at 20 days post-emergence inthese experiments.

The results of these field trials indicate that all strains used in thistrial have an overall positive effect on soy yields, but that thiseffect is most pronounced with NLS0017 applied as either a foliar orin-furrow treatment. Higher PPFM application rates generally resulted ingreater yield increases. This trend further supports the beneficialeffect of PPFM bacteria on soy yields and suggests that larger PPFMpopulations increase this effect.

Individual location data indicate that PPFMs generally have a beneficialeffect on soy yield. RFR, the single site where PPFMs did not positivelyinfluence soy yield, had particularly high overall yields. This couldindicate that RFR had an ‘ideal’ yield environment and suggests that thePPFM treatments used in these experiments offered yield protection andincreased yield in the presence of various biotic and/or abioticstressors at other locations but did not significantly affect yieldunder ideal growth conditions at RFR.

REFERENCES

-   1. Abanda-Nkpwatt, D., M. Musch, J. Tschiersch, M. Boettner, and W.    Schwab. 2006. Molecular interaction between Methylobacterium    extorquens and seedlings: growth promotion, methanol consumption,    and localization of the methanol emission site. J. Exp. Bot. 57:    4025-4032.-   2. Broekaert W F, Terras F R, Cammue B P, Vanderleyden J (1990) An    automated quantitative assay for fungal growth inhibition. FEMS    Microbiology Letters 69: 55-60.-   3. Cao, Y-R, Wang, Q., Jin, R-X., Tang, S-K., He, W-X., Lai, H-X,    Xu, L-H., and C-L Jiang. 2011. Methylobacterium soli sp. nov. a    methanol-utilizing bacterium isolated from the forest soil. Antonie    van Leeuwenhoek (2011) 99:629-634.-   4. Corpe, W. A., and D. V. Basile. 1982. Methanol-utilizing bacteria    associated with green plants. Devel. Industr. Microbiol. 23:    483-493.-   5. Corpe, W. A., and S. Rheem. 1989. Ecology of the methylotrophic    bacteria on living leaf surfaces. FEMS Microbiol. Ecol. 62: 243-250.-   6. Green, P. N. 2005. Methylobacterium. In Brenner, D. J., N. R.    Krieg, and J. T. Staley (eds.). “Bergey's Manual of Systematic    Bacteriology. Volume two, The Proteobacteria. Part C, The alpha-,    beta-, delta-, and epsilonproteobacteria.” Second edition. Springer,    New York. Pages 567-571.-   7. Green, P. N. 2006. Methylobacterium. In Dworkin, M., S.    Falkow, E. Rosenberg, K.-H. Schleifer, and E. Stackebrandt (eds.).    “The Prokaryotes. A Handbook on the Biology of Bacteria. Volume 5.    Proteobacteria: Alpha and Beta Subclasses.” Third edition. Springer,    New York. Pages 257-265.-   8. Holland, M. A. 1997. Methylobacterium and plants. Recent. Res.    Devel. in Plant Physiol. 1: 207-213.-   9. Holland, M. A., and J. C. Polacco. 1994. PPFMs and other covert    contaminants: Is there more to plant physiology than just plant?    Annu Rev. Plant Physiol. Plant Mol. Biol. 45: 197-209.-   10. Kutschera, U. 2007. Plant-associated methylobacteria as    co-evolved phytosymbionts. A hypothesis. Plant Signal Behav. 2:    74-78.-   11. Lidstrom, M. E. 2006. Aerobic methylotrophic prokaryotes. In    Dworkin, M., S. Falkow, E. Rosenberg, K.-H. Schleifer, and E.    Stackebrandt (eds.). “The Prokaryotes. A Handbook on the Biology of    Bacteria. Volume 2. Ecophysiology and biochemistry.” Third edition.    Springer, New York. Pages 618-634.-   12. Madhaiyan, M., S. Poonguzhali, H. S. Lee, K. Hari, S. P.    Sundaram, and T. M. Sa. 2005. Pink-pigmented facultative    methylotrophic bacteria accelerate germination, growth and yield of    sugarcane clone Co86032 (Saccharum officinarum L.) Biol. Fertil.    Soils 41: 350-358.-   13. Madhaiyan, M., S. Poonguzhali, M. Senthilkumar, S. Seshadri, H.    Chung, J. Yang, S. Sundaram, and T. Sa. 2004. Growth promotion and    induction of systemic resistance in rice cultivar C0-47 (Oryza    sativa L.) by Methylobacterium spp. Bot. Bull. Acad. Sin. 45:    315-324.-   14. Madhaiyan, M., S. Poonguzhali, and T. Sa. 2007. Influence of    plant species and environmental conditions on epiphytic and    endophytic pink-pigmented facultative methylotrophic bacterial    populations associated with field-grown rice cultivars. J Microbiol    Biotechnol. 2007 October; 17(10): 1645-54.-   15. Stanier, R. Y., N. J. Palleroni, and M. Doudoroff. 1966. The    aerobic pseudomonads: A taxonomic study. J. Gen. Microbiol. 43:    159-271.-   16. Sy, A., Giraud, E., Jourand, P., Garcia, N., Willems, A., De    Lajudie, P., Prin, Y., Neyra, M., Gillis, M., Boivin-Masson, C., and    Dreyfus, B. 2001. Methylotrophic Methylobacterium Bacteria Nodulate    and Fix Nitrogen in Symbiosis with Legumes. Jour. Bacteriol.    183(1):214-220,-   17. Sy, A., A. C. J. Timmers, C. Knief, and J. A. Vorholt. 2005.    Methylotrophic metabolism is advantageous for Methylobacterium    extorquens during colonization of Medicago truncatula under    competitive conditions. Appl. Environ. Microbiol. 71: 7245-7252.18.    Vogel, H. J., and D. M. Bonner. 1956. Acetylornithinase of    Escherichia coli: Partial purification and some properties. J. Biol.    Chem. 218: 97-106.-   18. Vogel, H. J. 1956. A convenient growth medium for Neurospora    (Medium N). Microbial Genet Bull 13: 42-43-   19. Whittenbury, R., S. L. Davies, and J. F. Wilkinson. 1970.    Enrichment, isolation and some properties of methane-utilizing    bacteria. J. Gen. Microbiol. 61: 205-218.

Having illustrated and described the principles of the presentinvention, it should be apparent to persons skilled in the art that theinvention can be modified in arrangement and detail without departingfrom such principles.

Although the materials and methods of this invention have been describedin terms of various embodiments and illustrative examples, it will beapparent to those of skill in the art that variations can be applied tothe materials and methods described herein without departing from theconcept, spirit and scope of the invention. All such similar substitutesand modifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

What is claimed is:
 1. A method for improving soybean plant yield thatcomprises: (a) applying a composition comprising a Methylobacterium sp.to a soybean seed, or to a soybean plant or part thereof at a first timeof about the VE to about the V4 stage of development; and/or at a secondtime about the V5 to about the R6 stage of development; wherein thecomposition comprises: (i) a solid substance with the Methylobacteriumgrown thereon and adhered thereto; (ii) an emulsion having theMethylobacterium grown therein; or (iii) a Methylobacterium isolateISO02 (NRRL B-50930), ISO03 (NRRL B-50931), ISO09 (NRRL B-50937), ISO10(NRRL B-50938), or a derivative thereof and an agriculturally acceptableadjuvant, excipient, or combination thereof; and, (b) growing thesoybean plant to maturity, thereby improving yield of the soybean plant.2. The method of claim 1, wherein the composition is applied in saidfirst time at the VE to V2 stage of development, at the V2 to the V3stage of development, at the V3 to V4 stage of development, or at aboutthe V3 stage of development; and/or wherein the composition is appliedat the V6 to the R1 stage of development, at the R1 to R2 stage ofdevelopment, or at about the R1 stage of development.
 3. The method ofclaim 1, wherein the composition comprises: (i) a solid substance withthe Methylobacterium grown thereon and adhered thereto.
 4. The method ofclaim 3, wherein the composition comprises the Methylobacterium sp. at atiter of about 1×10⁶ CFU/gm to about 1×10¹⁴ CFU/gm for a solidcomposition or at a titer of about 1×10⁶ CFU/mL to about 1×10¹¹ CFU/mLfor a liquid composition containing the solid substance or for theemulsion.
 5. The method of claim 1, wherein the composition comprisesthe solid substance or the emulsion and wherein the Methylobacterium sp.is ISO02 (NRRL B-50930), ISO03 (NRRL B-50931), ISO09 (NRRL B-50937),ISO10 (NRRL B-50938), or a derivative thereof.
 6. The method of claim 1,wherein the Methylobacterium sp. is a glyphosate resistant orglufosinate resistant derivative of ISO02 (NRRL B-50930), ISO03 (NRRLB-50931), ISO09 (NRRL B-50937), or ISO10 (NRRL B-50938).
 7. The methodof claim 1, wherein the applied composition coats or partially coats thesoybean seed or the soybean plant or a part thereof.
 8. The method ofclaim 1, wherein the composition is applied to foliage of the soybeanplant.
 9. The method of claim 1, wherein the Methylobacterium sp. isselected for glyphosate resistance or glufosinate resistance.
 10. Themethod of claim 9, wherein the Methylobacterium sp. is selected forglyphosate resistance, wherein the soybean plant is a glyphosatetolerant soybean plant, and wherein a formulation containing glyphosateis also applied at about the V2 to about the V4 stage of soybean plantdevelopment.
 11. The method of claim 1, further comprising the step ofharvesting seed from the mature soybean plant.
 12. The method of claim11, wherein yield of harvested seed is increased in comparison to yieldof harvested seed obtained from a control soybean plant that did notreceive an application of the Methylobacterium sp.
 13. A soybean plantor soybean plant part that is coated or partially coated with acomposition comprising a Methylobacterium sp. isolate selected from thegroup consisting of ISO02 (NRRL B-50930), ISO03 (NRRL B-50931), ISO09(NRRL B-50937), ISO10 (NRRL B-50938), and derivatives thereof.
 14. Thesoybean plant or soybean plant part of claim 13, wherein the compositioncomprises: (i) a solid substance with the Methylobacterium grown thereonand adhered thereto; or (ii) an emulsion having the Methylobacteriumgrown therein.
 15. The soybean plant or soybean plant part of claim 13,wherein the composition comprises the Methylobacterium sp. at a titer ofabout 1×10⁶ CFU/gm to about 1×10¹⁴ CFU/gm for a solid composition or ata titer of about 1×10⁶ CFU/mL to about 1×10¹¹ CFU/mL for a liquidcomposition containing the solid substance or for the emulsion.
 16. Thesoybean plant or soybean plant part of claim 13, wherein theMethylobacterium sp. is Methylobacterium isolate ISO09 (NRRL B-50937),ISO10 (NRRL B-50938), or a derivative thereof.
 17. The soybean plant orsoybean plant part of claim 13, wherein the soybean plant part isselected from the group consisting of a seed, a stem, a flower, a leaf,a petiole, a pod, and an axillary bud.
 18. A method for improvingsoybean plant yield that comprises: (a) applying a compositioncomprising a Methylobacterium sp. to a soybean seed or to a soybeanplant at about the VE to about the V5 stage of soybean plantdevelopment, wherein the composition comprises: (i) a solid substancewith the Methylobacterium grown thereon and adhered thereto; (ii) anemulsion having the Methylobacterium grown therein; or (iii) aMethylobacterium isolate ISO02 (NRRL B-50930), ISO03 (NRRL B-50931),ISO09 (NRRL B-50937), ISO10 (NRRL B-50938), or a derivative thereof andan agriculturally acceptable adjuvant, excipient, or combinationthereof; and, (b) growing the soybean plant to maturity, therebyimproving yield of the soybean plant.
 19. The method of claim 18,wherein the composition is applied at about the V2 to about the V3 stageof development, about the V3 to about the V4 stage of development, orabout the V3 stage of development.
 20. The method of claim 18, whereinthe composition comprises the Methylobacterium sp. at a titer of about1×10⁶ CFU/gm to about 1×10¹⁴ CFU/gm for a solid composition or at atiter of about 1×10⁶ CFU/mL to about 1×10¹¹ CFU/mL for a liquidcomposition containing the solid substance or for the emulsion.
 21. Themethod of claim 18, wherein the composition comprises the solidsubstance or the emulsion and wherein the Methylobacterium sp. isselected from the group consisting of ISO02 (NRRL B-50930), ISO03 (NRRLB-50931), ISO09 (NRRL B-50937, ISO10 (NRRL B-50938), and derivativesthereof.
 22. The method of claim 18, wherein the Methylobacterium sp. isMethylobacterium isolate is ISO09 (NRRL B-50937), ISO10 (NRRL B-50938),or a derivative thereof.
 23. The method of claim 18, wherein theMethylobacterium isolate is selected for glyphosate resistance orglufosinate resistance.
 24. The method of claim 23, wherein the soybeanplant is a glyphosate tolerant soybean plant, wherein theMethylobacterium sp. is selected for glyphosate resistance, and whereina formulation containing glyphosate is also applied at about the V2 toabout the V4 stage of soybean plant development.
 25. The method of claim18, wherein the method further comprises harvesting seed from the maturesoybean plant.
 26. The method of claim 25, wherein yield of harvestedseed is increased in comparison to yield of harvested seed obtained froma control soybean plant that did not receive an application of theMethylobacterium sp.